Subset (5)

F1000Journal · 8.11k rows

Split (1)

data · 8.11k rows

link stringlengths 41 45	date stringlengths 9 9	paper dict	reviews listlengths 1 6	version int64 1 5	main stringlengths 38 42
https://f1000research.com/articles/13-863/v1	01 Aug 24	{ "type": "Case Report", "title": "Case Report: An unusual case of severe autoimmune hemolytic anemia in untreated hepatitis C viral infection", "authors": [ "H Debbabi", "A Chakroun", "H Elloumi", "H Yacoub", "S Ben Azouz", "R Marouani", "H Debbabi", "A Chakroun", "H Elloumi", "S Ben Azouz", "R Marouani" ], "abstract": "Patients with hepatitis C share a variety of anemia, including bleeding, nutritional deficiencies, and anemia of chronic disease. Autoimmune hemolytic anemia (AIHA) is usually reported with interferon-alpha and/or ribavirin treatment in hepatitis C virus (HCV) infections. We report an unusual case of AIHA occurring in a49-year-old patient with untreated hepatitis C infection who presented with functional anemia syndrome. Monospecific direct antiglobulin test showed the presence of anti-complement (C3d++). Cold agglutinin titer was 1/16 at 4°C. The patient improved following blood transfusion, steroid therapy and direct acting antivirals.", "keywords": [ "Cold agglutinin- autoimmune hemolytic anemia- Chronic hepatitis C" ], "content": "Introduction\n\nChronic hepatitis C virus (HCV) infection is widely recognized as a cause of extrahepatic manifestations, including autoimmune disorders.1–4 Autoimmune hemolytic anemia (AIHA) is rare. Warm AIHA with HCV infection in treatment-naïve patients has been described either during interferon treatment or in treatment-naive patients.4–7 However, the association between AIHA due to cold agglutinin and untreated hepatitis C seems to be a rare situation, to the best of our knowledge only two cases have been reported.8,9 Herein, we present a case of HVC infection complicated by cold AIHA.\n\n\nCase report\n\nA 49-year-old man with a history of HVC diagnosed two months ago, presented with dyspnea and pallor. HCV belonged to the type 1a genotype and the viral load was 1.6 106 UI/ml. Fibroscan showed F2 hepatic fibrosis stage. No evidence of recent drug use was found. On admission, the patient was pale, polypneic, and tachycardic with mild hepatomegaly. The patient did not have splenomegaly or lymphadenopathy. Laboratory tests showed severe anemia with a low hemoglobin (Hb) level (4.2 g/dl), macrocytosis (MCV 120 fL), reticulocytosis (5.2%), and normal white blood cell and platelet counts. A peripheral blood smear showed anisocytosis and polychromasia with rare schizocytes. Hemolysis workup revealed elevated indirect bilirubin (84 μmol/l), high lactate dehydrogenase (LDH) (580 U/L), and low haptoglobin concentration (0.15 g/l). These findings were consistent with the diagnosis of hemolytic anemia.\n\nThe presence of anti-complement C3d++ was confirmed using a monospecific direct antiglobulin test (DAT- Low Ionic Strength Solution). Anti-immunoglobulin G antibodies were absent. Screening of the eluate was negative, and the cold agglutinin titer was 1/16 at 4°C. Hemoglobin electrophoresis results were normal. A bone marrow biopsy was performed to elucidate the underlying etiology of the patient’s AIHA and showed erythroid hyperplasia with no evidence of tumor lymphoma. Further investigations, including anti-smooth muscle, antinuclear antibodies, and anti-DNA antibodies, were negative. However, type III mixed cryoglobulinemia was also observed. Thoracic-abdominal computed tomography (CT) indicated that the underlying malignancy was ruled out. Serology results for hepatitis B and human immunodeficiency virus (HIV) were also negative. Based on the above findings, a diagnosis of AIHA due to cold agglutinin complicating untreated chronic hepatitis C was made. The patient required repeated packed RBC transfusions on admission. When the diagnosis of AIHA was confirmed, the patient received corticosteroid therapy (1.5 mg/kg/day) for six weeks. No additional blood transfusion was needed because a remarkable improvement in the Hb level was obtained (9.8 g/dl). After three months, the patient started HCV treatment with oral antiviral therapy. During the follow-up, the patient’s hematologic disorders were completely renormalized.\n\n\nDiscussion\n\nHCV-related immune disorders are frequent secondary to the activation of the immune system.\n\nHCV lymphotropism determines dysregulation of the immune system, facilitating clonal B-lymphocyte expansion and autoantibody production.10\n\nThis state represents a trigger in the pathogenesis of virus-related immune disorders both in systemic autoimmune diseases (Sjögren syndrome, rheumatoid arthritis, etc.) and in organ-specific autoimmune diseases (diabetes mellitus, thyroid disorders, etc.).11\n\nA wide variety of hematologic disorders associated with HCV, such as anemia, neutropenia, and thrombocytopenia, are well documented and are usually associated with interferon (IFN) and ribavarin therapy.2,12–14 It has been suggested that AIHA observed in untreated HCV infection occurs especially in cirrhotic patients, who have a higher prevalence of autoimmune diseases and respond well to corticosteroid therapy.12 However, HCV-related AIHA is uncommon in treatment-naive patients.4\n\nMost cases previously reported in the literature describe warm AIHA with no history of autoimmune disorders.14–19 Anemia was not associated with any other hematological disorders with a good response to steroid therapy.\n\nHowever, the association between AIHA due to cold agglutinin and untreated hepatitis C seems rare. To the best of our knowledge, only two cases have been reported.8,9 In the latter, cold agglutinin-mediated hemolytic anemia occurred in 72- and 74-year-old men who were positive for type III mixed cryoglobulinemia, as in our patient. Mixed cryoglobulinemia is the most documented extrahepatic manifestation of HCV infection.20\n\nA common hypothesis for HCV-related cryoglobulinemia is chronic antigen stimulation of the humoral immune system, with clonal b-lymphocyte expansion producing circulating immunocomplexes. It may also facilitate the development of autoimmune diseases and lymphoproliferative disorders. However, in our patient, as well as in the two published cases, there was no evidence of lymphoproliferative disorder.9\n\nOur patient was diagnosed with cold agglutinins AIHA, including hemolytic anemia, reticulocytosis, elevated lactate dehydrogenase, hyperbilirubinemia, positive direct antiglobulin test (DAT), monospecific DAT positive for C3d, and negative for IgG and cold agglutinin titers ≥ 64. In our patient, the thermal amplitude, which is the highest temperature at which the antibody reacts with the antigen, at 4°C was useful for diagnosis, considering the cold agglutinin titer < 64.21\n\nCold agglutinins can adhere to red blood cells at low temperatures (complement activation). C3b-coated erythrocytes are removed by macrophages.22\n\nSubsequently, it is imperative to rule out potential etiologies of cold agglutinin-induced AIHA, including medication side effects, systemic lupus erythematosus, and malignancy. In our patient and in two similar published cases, no etiology was found, except for HCV infection.\n\nPathogenesis of AIHA induced by infectious agents is still not well understood. Inflammatory state may explain this rare condition.23,24\n\nDifferent treatment modalities are available for AIHA: corticosteroids, anti-CD20 (rituximab), Intravenous immunoglobulin (IVIG), and splenectomy. However, in cold AIHA, steroids are less effect in patients with cold AIHA compared with patients with warm AIHA.14,19,25 Our patient was a good responder to corticosteroids.\n\nSteroids do not seem to show good results in all cases.8 In this situation, anti-CD20 or IVIG may be considered.26 However, it is important to note that the concomitant use of HCV antiretroviral drugs could explain the good response to corticosteroid therapy observed in our patient.\n\n\nConclusions\n\nAIHA due to cold agglutinin is a very uncommon hematologic manifestation of HCV infection, particularly in treatment-naive patients. HCV seems to be mysterious, and further research is needed to elucidate the mechanisms of some related HCV autoimmune manifestations.\n\n\nConsent\n\nPatient gave an informed written consent to publish details regarding the case summary and its use in medical publications.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nCanavese C, Hollò Z, Ciccone G, et al.: Extrahepatic immunological manifestations of hepatitis C virus in dialysis patients. J. Nephrol. 2000; 13(5): 352–359. PubMed Abstract\n\nBayraktar Y, Bayraktar M, Gurakar A, et al.: A comparison of the prevalence of autoantibodies in individuals with chronic hepatitis C and those with autoimmune hepatitis: the role of interferon in the development of autoimmune diseases. Hepato-Gastroenterology. 1997; 44(14): 417–425. PubMed Abstract\n\nClifford BD, Donahue D, Smith L, et al.: High prevalence of serological markers of autoimmunity in patients with chronic hepatitis C. Hepatology. 1995; 21(3): 613–619. PubMed Abstract\n\nBianco C, Coluccio E, Prati D, et al.: Diagnosis and Management of Autoimmune Hemolytic Anemia in Patients with Liver and Bowel Disorders. J. Clin. Med. 2021 22; 10(3): 423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasseri RJ, Schmidt MT, Basseri B: Autoimmunehemolyticanemia in treatment-naivechronichepatitis C infection: a case report and review of literature. Clin. J. Gastroenterol. 2010; 3(5): 237–242. PubMed Abstract \| Publisher Full Text\n\nOliveira TL, Caetano AZ, Belem JM, et al.: Interferon-α induced psoriatic arthritis and autoimmune hemolytic anemia during chronic hepatitis C treatment. Acta Reumatol. Port. 2014; 39(4): 327–330. PubMed Abstract\n\nCauli C, Serra G, Chessa L, et al.: Severe autoimmune hemolytic anemia in a patient with chronic hepatitis C during treatment with peginterferon alfa-2a and ribavirin. Haematologica. 2006; 91: ECR26. PubMed Abstract\n\nEtienne A, Gayet S, Vidal F, et al.: Severe hemolytic anemia due to cold agglutinin complicating untreated chronic hepatitis C: Efficacy and safety of anti-CD20 (rituximab) treatment: Brief Report: Hemolytic Anemia Due to Cold Agglutinin in Untreated Hepatitis C. Am. J. Hematol. 2004 Apr; 75(4): 243–245. PubMed Abstract \| Publisher Full Text\n\nRuivard M, Tridon A, Quainon F, et al.: Agglutinines froides et cryoglobulinémie chez un patient avec une hépatite C [Cold agglutinins and cryoglobulinemia in a patient with hepatitis C]. Presse Med. 1996; 25(32): 1548–1549. French. PubMed Abstract\n\nZignego AL, Ferri C, Pileri SA, et al.: Extrahepatic manifestations of Hepatitis C Virus infection: a general overview and guidelines for a clinical approach. Dig. Liver Dis. 2007; 39: 2–17. PubMed Abstract \| Publisher Full Text\n\nCalvaruso V, Craxì A: Immunological alterations in hepatitis C virus infection. World J. Gastroenterol. 2013; 19: 8916–8923. Publisher Full Text\n\nReau N, Hadziyannis SJ, Messinger D, et al.: Early predictors of anemia in patients withhepatitis C genotype 1 treated with peginterferon alfa-2a (40KD) plus ribavirin. Am. J. Gastroenterol. 2008; 103(8): 1981–1988. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavidovitz Y, Halpern Z, Vardi J, et al.: Pure Red Cell Aplasia Responsive to Interferon-α in a Patient with Hepatitis C Virus Infection. Acta Haematol. 1998; 100(4): 213–215. Publisher Full Text\n\nSrinivasan R: Autoimmune Hemolytic Anemia in Treatment-naïve Chronic Hepatitis C Infection. J. Clin. Gastroenterol. 2001; 32(3): 245–247. PubMed Abstract \| Publisher Full Text\n\nRamos-Casals M, García-Carrasco M, López-Medrano F, et al.: Severe Autoimmune Cytopenias in Treatment-naïve Hepatitis C Virus Infection: Clinical Description of 35 Cases. Medicine (Baltimore). 2003; 82(2): 87–96. PubMed Abstract \| Publisher Full Text\n\nElhajj II, Sharara AI, Taher AT: Chronic hepatitis C associated with Coombs-positive hemolytic anemia. Hematol. J. 2004; 5(4): 364–366. PubMed Abstract \| Publisher Full Text\n\nChao TC, Chen CY, Yang YH, et al.: Chronic hepatitis C virus infection associated with primary warm-type autoimmune hemolytic anemia. J. Clin. Gastroenterol. 2001 Sep; 33(3): 232–233. PubMed Abstract \| Publisher Full Text\n\nMoccia F, Tognoni E, Boccaccio P: Autoimmune hemolytic anemia in chronic hepatitis C virus infection: an unusual extrahepatic autoimmune manifestation. Ann. Ital. Med. Int. 2001; 16(4): 256–259. PubMed Abstract\n\nEspinosa BerenguelJ L, Muñoz Sánchez JA: Autoimmune hemolytic anemia and post hepatitis-C liver cirrhosis. An. Med. Interna. 1997; 14(11): 583–584.\n\nFerri C, Greco F, Longombardo G, et al.: Association between hepatitis C virus and mixed cryoglobulinemia [see comment]. Clin. Exp. Rheumatol. 1991; 9: 621–624. PubMed Abstract\n\nJäger U, Barcellini W, Broome CM, et al.: Diagnosis and treatment of autoimmune hemolytic anemia in adults: Recommendations from the First International Consensus Meeting. Blood Rev. 2019; 41: 100648. Publisher Full Text\n\nJaffe CJ, Atkinson JP, Frank MM: The role of complement in the clearance of cold agglutinin-sensitized erythrocytes in man. J. Clin. Invest. 1976; 58: 942–949. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazarian G, Quinquenel A, Bellal M, et al.: Autoimmune haemolytic anaemia associated with COVID-19 infection. Br. J. Haematol. 2020; 190: 29–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrabhu R, Bhaskaran R, Shenoy V, et al.: Clinical characteristics and treatment outcomes of primary autoimmune hemolytic anemia: a single center study from South India. Blood Res. 2016; 51(2): 88–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerentsen S: How I manage cold agglutinin disease: Review. Br. J. Haematol. 2011; 153(3): 309–317. PubMed Abstract \| Publisher Full Text\n\nBerentsen S: New insights in the pathogenesis and therapy of cold agglutinin-mediated autoimmune hemolytic anemia. Front. Immunol. 2020; 11: 590. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "324843", "date": "01 Oct 2024", "name": "Madhumita Premkumar", "expertise": [ "Reviewer Expertise Hepatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 single report of AIHA occurring in a 49-year-old patient with untreated hepatitis C infection who presented with 'functional' anemia syndrome.\n\n1. Please advise if it was appropriate to start steroids without the cover of direct acting antiviral agents (DAAs) given that the patient has f2 fibrosis and could worsen. 2. How were other causes of hemolytic anemia ruled out? What is a functional anemia syndrome? 3. What was the regimen of DAA prescribed? Did the patient attain SVR-12? 4. Was a liver biopsy done? 5. Change the statement 'HCV seems to be mysterious, and further research is needed to elucidate the mechanisms of some related HCV autoimmune manifestations.' There is no new information in this report to suggest mystery.\nPlease see the additional references[ref 1][ref 2] and [ref 3].\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "324838", "date": "14 Oct 2024", "name": "Batbold Batsaikhan", "expertise": [ "Reviewer Expertise liver fibrosis", "HCV", "HCC" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is well written and included all necessary patient's condition. Also it is well described extrahepatic manifestations such as cryoglobulinemia. It is informative for practitioners that chronic HCV infection can be related with cold autoimmune hemolytic anemia. Authors may address SVR condition after HCV treatment, it is sure that patient responded the treatment.\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/13-863
https://f1000research.com/articles/13-862/v1	01 Aug 24	{ "type": "Systematic Review", "title": "The use of guest speakers in higher education and implications for pharmaceutical teaching: a systematic review of literature", "authors": [ "ALI ALSHAHRANI" ], "abstract": "Background The purpose of this paper was to systematically review the current literature on the use of guest speakers in higher education teaching and evaluate the implications of the findings for teaching in pharmaceutical teaching.\n\nMethods A comprehensive search of different academic databases for literature on the use of guest speakers in higher education teaching was conducted and articles meeting the inclusion criteria were included in the analysis. A total of 15 articles were used in this systematic review.\n\nResults The use of guest speakers has critical benefits including promoting better learning, good career preparedness, positive learning outcomes, and creating an environment where learners have the opportunity to perceive real-world scenarios from the experiences of the guest speakers and thus be better prepared for the job market. With the increasing importance of the role of guest speakers in higher education learning, researchers have been engaging in coming up with standards and best practices for enhancing the effectiveness of the use of guest speakers in learning. The use of guest speakers in the teaching of pharmacy can enhance positive health outcomes by allowing students to have a better view of the situations they will deal with in their careers.\n\nConclusion The use of guest speakers can have major positive outcomes in promoting effectiveness in learning among students of pharmacy. Professors teaching pharmacy courses have the opportunity to benefit from various tools and resources that academic researchers have developed for guiding the process of the use of guest speakers in pharmacy teaching.", "keywords": [ "Guest Speakers", "Higher Education", "Pharmaceutical Teaching", "Education", "Pharmacy Education." ], "content": "Introduction\n\nGuest speakers have become a common feature in college and university teaching as they bring a wealth of knowledge and experience to the classroom. This experience has been useful in enhancing the process of learning for students (Zheng et al., 2018). Guest speakers can be individuals who have expertise in their fieldas well as who have relevant industry experience (Zorek et al., 2011). The expertise knowledge and industry experience are both instrumental assets for individuals serving as guest speakers in that they understand aspects that might be beneficial to learners in the long run, especially once they join the job market. The experience of these individuals has provided valuable insights into real-world scenarios and current trends and practices as well as case studies for improved learning in the higher education sector (Sortedahl & Imhoff, 2016). Guest speakers come from different backgrounds, cultures, and experiences, and, for this reason, can offer a unique perspective on various topics and bring diversity into the classroom (Merle & Craig, 2016). Incorporating guest speakers into college and university teaching serves as a means of networking for students, allowing them the opportunity to interact with professionals in their field of study and potentially form connections that can help them in their future careers (Jablon-Roberts & McCracken, 2023; Sage, 2013; Zheng et al., 2018). Noting the inclusive and participatory environment for learning that is necessary for learners in higher education, Sage (2013) explained that guest speakers provide an opportunity for students to engage in active learning as they able to ask questions, participate in discussions within the classroom with their peers and instructors, and apply the concepts learned in class to real-world situations.\n\nCurrent research shows that the concept of guest speakers and its application in enhancing the process learning in higher education has been increasingly adopted and utilized across different academic fields from nursing, tourism, natural sciences, and even in the textile classroom (Riebe et al., 2013; Rockich-Winston et al., 2018). In all ofthese environments, guest speakers have enriched the process of learning for students in significant ways. For example, McCleary and Weaver (2008) explained that in the hospitality and tourism industry, guest speakers who are experts in their respective fields provide a deeper understanding of various destinations and cultures, helping to broaden the perspective of tourism students (Metrejean et al., 2002). In the nursing field, guest speakers who are practicing nurses or healthcare professionals have been utilized by nursing course instructors to share their experiences and knowledge, providing valuable insights and guidance to their students (Zou et al., 2019). This exposure to the knowledge of these industry professionals not only helps students to understand the practical aspects of the profession but also gives them an opportunity to network with professionals in the field (Dalakas, 2016).\n\nIn natural sciences classroom environments, guest lectures by scientists or researchers have proven vital in providing students in these classes with a more comprehensive understanding of complex scientific concepts taught at the higher education level (Tenenberg, 2009). With the high need for continuous research in the natural sciences field, equipping students with this knowledge and experience from guest speakers has been a beneficial tool which allows them to gain a better appreciation of the subject matter and inspires them to pursue further research (Hemphill and Hemphill, 2007; Jablon-Roberts & McCracken, 2022). Even in the textile classroom, the use of guest speakers such as fashion designers or textile industry experts has empowered students with valuable insights into the industry and its trends, as well as practical knowledge on design and production techniques which are essential for success in their future careers (Jablon-Roberts & McCracken, 2023).\n\nNoting the vital importance of the role of guest speakers in diverse classrooms and courses at the higher education level, attention is needed to guide the understanding of how the use of these industry and field experts can impact the process of learning and student perceptions regarding the use of guest speakers on their future careers. The objective of this study was to conduct a systematic review of current literature on the use of guest speakers within the classroom environment at the higher education level and evaluate the implications that this may have for the process of learning and teaching in the pharmaceutical industry.\n\n\nMethods\n\nThe entire design of the methodology for this study was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) principles and directions for developing a systematic review of literature (Page et al., 2021).\n\nThe search for credible studies on the use of guest speakers in higher education was conducted via five different databases: Medline, BMC, ProQuest, EBSCOhost, and Google Scholar. The primary search for articles included all possible studies published between 2016 and May 2023, and those which directly addressed the topic of use of guest speakers in higher education. Special attention was given to articles specifically on the use of guest speakers in the teaching of pharmacy. A detailed search of the selected academic databases was carried out using the following keywords and phrases, and in some instances a combination of the phrases: “guest speaker in higher education,” “guest speakers in pharmacy classes,” “use of guest speakers in college teaching,” “use of guest speakers in university teaching,” “guest speakers in classrooms,” and “the impact of guest speakers in higher education.” Considering the limited number of studies that emerged directly on the use of guest speakers in pharmacy teaching, the researcher also considered and included studies that examinedthe general use of guest speakers in higher education teaching as the content proved relevant for informing the conclusions of the current study.\n\nThe number of records of studies obtained from each of the databases is shown in a flow diagram in Figure 1. The screening was a two-step process in which first, the abstracts and titles of the studies were screened for suitability of inclusion in the study. In the second stage of the screening, entire documents were assessed and those which were suitable were included. In the exclusion criteria, all initially selected studies that were found to be non-peer reviewed or not directly related to the use of guest speakers in higher education were excluded. During the final screening, the author evaluated the remaining studies for content similarity as well as content that was not directly conclusive on the impact of the use of guest speakers in higher education (Figure 1).\n\n\nResults\n\nThe flow chart diagram in Figure 1 captures the study selection process for this systematic review. The inclusion and exclusion criteria for the studies in this systematic review are reflected.\n\nAll the 15 included studies directly covered concepts that the author determined to be vital for guiding the understanding of how the use of guest speakers can impact the learning process higher education and how it can be impactful to students in the pharmacy class. A total of 9 (60.0%) studies from those encompassed the use of guest speakers in higher education at both the college and university levels. Five (33.3%) of the remaining sevenstudiesaddressed specific areas in higher education, including tourism, nursing, language classes, mass communication, and textiles. One of thestudiesconsisted of aspects such as approaches and models used by professors and instructors within institutions of higher learning for effective use of guest speakers in the learning process at higher education. The last study examined the legal aspects as they pertain to the use of guest speakers in higher education. Most of the studies were of qualitative design, while several others were quantitative with some being literature reviews on the specific areas. Four (26.7%) of the studies were quantitative, seven (46.7%) were qualitative, three (20.0%) were literature reviews, and one (6.3%) was an online survey.\n\nThe author systematically assessed each of the included studies for possible bias. The bias assessment encompassed several distinct facets, including the evaluation of potential bias in the selection of studies for inclusion, scrutiny of the foundations of individual research studies, verification of authorship to establish that the study selection and inclusion were notinfluencedby the author's potential knowledge or associations with the authors of said studies, and an assessment of overall credibility. The researcher ensured that each of the studies first selected was comprehensively screened under the strict inclusion and exclusion criteria, primarily to ensure that all studies that could inform the current systematic review were included in the review.\n\nResults of the individual studies reviewed\n\nThe results of the individual studies reviewed are shown in Table 1(Extended data), included separately in another file.\n\nSummary of the results\n\nThe summarized results from various studies on the use of guest speakers in education highlight several key findings. In the realm of student perceptions, multiple studies, including Jablon-Roberts and McCracken (2022, 2023), emphasized the positive impact of guest speakers on student learning experiences. These sessions were particularly helpful during challenging situations like lockdowns (Jablon-Roberts & McCracken, 2023) as well as enhancing overall student perceptions about the importance of guest speakers (Jablon-Roberts & McCracken, 2022).\n\nFurthermore, the effectiveness of guest speakers is contingent on their expertise, especially in specific industries like mass communication (Merle & Craig, 2016) and nursing (Zou et al., 2019). Integrative reviews and observational studies, such as those conducted by Sortedahl and Imhoff (2016) and McCleary and Weaver (2008) revealed that incorporating professionals from their respective fields as guest speakers significantly contributes to student learning, empowering them for success in their future careers. The studies also stressed the importance of proper planning, organization, and legal considerations. Addressing legal aspects as outlined by Sage (2013) is crucial, particularly in higher education settings. Additionally, student-centered approaches, group engagement (Li & Guo, 2015), and innovative session formats (Dalakas, 2016) were emphasized to enhance student participation and understanding during guest speaker sessions. The research underscores that guest speakers play a pivotal role in enhancing the learning processand also bridging the gap between theoretical knowledge and real-world applications. Their impact is multifaceted, influencing student perceptions, engagement, and future career prospects, making them a valuable asset in educational contexts.\n\n\nDiscussion\n\nThe use of guest speakers has been widely accepted across different content areas and fields in higher education, and the literature shows that there are substantial benefits that arise—particularly to students—following the use of guest speakers. According to Jablon-Roberts and McCracken (2023), online guest speakers have been found to be beneficial inenhancing learning for students, especially in difficult situations like lengthy lockdowns such as those during the COVID-19 pandemic. For heightened results and benefits for students in the use of guest speakers, it is important to recruit experts in the specific area of the students’ future careers (Badia, 2015; Merle & Craig, 2016). Several tools have been developed to foster the effective use of guest speakers in higher education, including methods for contacting appropriate guest speakers (Wetzel, 2012) as well as regulations to govern the use of these speakers, especially in an online environment where instructors using virtual guest speakers must be aware of the provisions of Family Education Rights and Privacy Act of 1974. Across wide areas in higher education, the use of guest speakers has proven useful and it is important that instructors in the pharmacy field understand this need and focus on integrating this approach in teaching pharmacy students.\n\nWithin the pharmaceutical sector, effective teaching is mandatory as this is a field where graduates transition to serve society in the sensitive area of offering pharmaceutical services. Guest speakers can play a significant role in pharmaceutical teaching by providing students with valuable insights into the industry, current practices, and real-world scenarios. These speakers can be experts in their field or have relevant industry experience, and can offer students a unique perspective on the pharmaceutical industry. Guest speakers can share their experiences, discuss emerging trends, and provide insights into the latest research and developments in the pharmaceutical industry. They can also discuss regulatory frameworks, ethical considerations, and challenges that the industry faces, giving students a comprehensive understanding of the pharmaceutical industry beyond what is taught in the classroom. Incorporating guest speakers into pharmaceutical teaching can also provide students with networking opportunities, allowing them to make connections and potentially find internships or job opportunities in the industry. Furthermore, students can engage in active learning through discussions and Q&A sessions with guest speakers, gaining a deeper understanding of the industry and its challenges.\n\nThere is credible evidence in current literature that the use of guest speakers can have major positive outcomes in promoting effectiveness in learning among pharmacy students and making them well prepared to facediverse,real-world situations during practice. Professors teaching pharmacy courses have the opportunity of benefiting from various tools and resources that academic researchers have developed for guiding the process of using guest speakers in pharmacy teaching.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nThe dataset associated with this study are available in the figshare repository under the following DOI: https://doi.org/10.6084/m9.figshare.26314159.v1 (Alshahran, 2024).\n\nThis project contains following data set.\n\nTable 1: Summary of the Individual Studies Reviewed\n\nFigure 1: Flow of the studies selection and inclusion process\n\nPRISMA 2020 Checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nThe authors extend their appreciation on Taif University, Saudi Arabia, for supporting this work through project number (TU-DSPP-2024-256).\n\n\nReferences\n\nAbdallah A: Guest speakers and internationalization in higher education: a critical reflection of guest speakers in tourism programmers. The Business of Tourism. 2016; 17: 61–70. Publisher Full Text\n\nAlbirini A: Using technology, literature and guest speakers to raise the cultural awareness of Arabic language learners. The International Journal of Language Society and Culture. 2009; 28: 1–15. Reference Source\n\nBadia G: Listen up, everyone! conquering students' inattentiveness when you're the guest lecturer. Issues Sci. Technol. Librariansh. 2015. Publisher Full Text Reference Source\n\nDalakas V: Turning guest speakers' visits into active learning opportunities. Atl. Mark. J. 2016; 5(2): 93–103. Reference Source\n\nFarruggio P: Bilingual education: Using a virtual guest speaker and online discussion to expand Latino preservice teachers’ consciousness. Multicult. Educ. 2009; 17(1): 33–37. Reference Source\n\nHemphill LS, Hemphill HH: Evaluating the impact of guest speaker postings in online discussions. Br. J. Educ. Technol. 2007; 38(2): 287–293. Publisher Full Text\n\nJablon-Roberts S, McCracken A: Undergraduate student perceptions of industry guest speakers in the college classroom. Journal of the Scholarship of Teaching and Learning. 2022; 22(3): 76–88. Publisher Full Text\n\nJablon-Roberts S, McCracken A: Virtual guest speakers in textile and apparel courses: student experiences and expectations. Cloth. Text. Res. J. 2023; 41(1): 43–56. Publisher Full Text\n\nLi L, Guo R: A student-centered guest lecturing: A constructivism approach to promote student engagement. Journal of Instructional Pedagogies. 2015; 15: 1–7. Reference Source\n\nMccleary KW, Weaver PA: The effective use of guest speakers in the hospitality and tourism curriculum. J. Teach. Travel Tour. 2008; 8(4): 401–414. Publisher Full Text\n\nMcRee AL, Madsen N, Eisenberg ME: Guest speakers in school-based sexuality education. American Journal of Sexuality Education. 2014; 9(2): 205–218. Publisher Full Text\n\nMerle PF, Craig C: Be my guest: A survey of mass communication students' perception of guest speakers. Coll. Teach. 2016; 65(2): 41–49. Publisher Full Text\n\nMetrejean C, Pittman J, Zarzeski MT: Guest speakers: reflections on the role of accountants in the classroom. Acc. Educ. 2002; 11(4): 347–364. Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPercy C, Rehill J, Kashefpakdel E, et al..: Insights and inspiration: exploring the impact of guest speakers in schools, Education and Employers, London.2019. viewed 12 Sep 2023. Reference Source\n\nRiebe L, Sibson R, Roepen D, et al.: Impact of industry guest speakers on business students’ perceptions of employability skills development. Ind. High. Educ. 2013; 27(1): 55–66. Publisher Full Text\n\nRockich-Winston N, Train BC, Rudolph MJ, et al.: Faculty motivations to use active learning among pharmacy educators. Curr. Pharm. Teach. Learn. 2018; 10(3): 277–284. PubMed Abstract \| Publisher Full Text\n\nSage M: Distance guest speakers in online synchronous classrooms: Practical and legal considerations. J. Teach. Soc. Work. 2013; 33(4-5): 385–392. Publisher Full Text\n\nSortedahl CK, Imhoff A: Perspectives from the field: Bringing nurse leaders into the classroom. Nurs. Educ. Perspect. 2016; 37(2): 113–114. PubMed Abstract\n\nTenenberg J: The ultimate guest speaker: A model for educator/practitioner collaboration. J. Comput. Sci. Coll. 2009; 25(1): 123–129. Publisher Full Text\n\nWetzel RD: Contacting guest speakers. J. Phys. Educ. Recreat. Dance. 2012; 83(6): 51–53. Publisher Full Text\n\nZheng SL, Chen YS, Wang X, et al.: From the source: Student-centered guest lecturing in a chemical crystallography class. Journal of Applied Crystalography. 2018; 51(3): 909–914. Publisher Full Text\n\nZorek JA, Katz NL, Popovich NG: Guest speakers in a professional development seminar series. Am. J. Pharm. Educ. 2011; 75(2): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou P, Sun W, Hallowell SG, et al.: Use of guest speakers in nursing education: An integrative review of multidisciplinary literature. Advances in Medical E. 2019; 10: 175–189. Publisher Full Text\n\nAlshahran A: Data Table. figshare. 2024, 16 Temmuz. Publisher Full Text" }	[ { "id": "317908", "date": "18 Sep 2024", "name": "Wienta Diarsvitri", "expertise": [ "Reviewer Expertise 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\nThe topic is interesting and suitable for higher education in developing countries. Some parts of the manuscript should be improved: 1. In the introduction section, the author should add the research gaps as the rationale for conducting the systematic review. 2. In the methods section, the author should specify the inclusion criteria for selected literature, related to the chosen study design, and critical review of the literature. 3. In the table of the results section, the author should mention the criteria for judging whether the use of guest speakers benefits the students, guest speakers, and institution. 4. The discussion, conclusion, and abstract should follow the revised results. Good 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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) No", "responses": [] }, { "id": "324113", "date": "04 Dec 2024", "name": "Agustinus Bandur", "expertise": [ "Reviewer Expertise Educational Leadership and Management", "Research Methodology", "Higher Education Internationalization" ], "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 content of the paper is relevant in the context of higher education teaching and learning, with particular reference to the provision of guest lecturer. However, unless major revision is made, the current version of the paper has limitations as outlined in more detail below:\n1) The current version of the paper has lack of international relevance as it does not provide wider and deeper discussion with other relevant studies worldwide; 2) The paper has shown the importance of guest speaker in the 'Introduction' section, but it failed to address the gaps in the current existing literature and accordingly, the paper has no clear contribution to the current knowledge development. 3) The paper has a good method for writing the SLR by using PRISMA. However, it needs more detailed elaboration of the bibliometric results of analysis. It requires further in-depth explanations. 4) On the basis of this SLR, it needs to provide future research direction. This important aspect is absent in the paper, both at the discussion section and conclusion. 5) The conclusion need to be further develop in order to provide convincing concluding remarks on the basis of the results.\n\nI am more than happy to provide further review after the major revision of this SLR paper.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/13-862
https://f1000research.com/articles/13-859/v1	01 Aug 24	{ "type": "Research Article", "title": "Effect of a pragmatic lifestyle modification intervention on physical activity levels and body mass index among obese and overweight adolescents in Udupi, India: a cluster randomized trial", "authors": [ "prateek srivastav", "Vaishali K", "H Vinod Bhat", "Suzanne Broadbent", "prateek srivastav", "H Vinod Bhat", "Suzanne Broadbent" ], "abstract": "Background Determine the effects of a multifactorial lifestyle intervention on physical activity (PA), BMI and health-related quality of life (QoL) in obese and overweight adolescents.\n\nMethods Nine schools in India were clustered randomly in a 12-month study with students allocated to a multifactorial intervention (MFI), or exercise only (EX) or control (CON) group. Participants were adolescents aged 11-16 years (n=671). In the MFI group, adolescents and their parents received lifestyle education using a validated booklet combined with a PA intervention for school students. The EX group received school-based PA only; the CON group continued regular activities. Primary outcomes were PA levels measured with the PAQ-A, and BMI; the secondary outcome was health-related QoL. A linear regression statistical model was used to analyse time, group effects and interactions, with Bonferroni correction for within-group differences at baseline (T0) and at 12-weeks (T1) (post-intervention), 6-month (T2) and 12-month (T3) follow-ups.\n\nResults Significant time and group effects observed for all groups with PA scores (p<0.001), with MFI group having largest increase in PA; with BMI (p<0.001) and MFI showing the least gain in BMI; and HRQOL (p<0.001), with MFI group showing greatest improvement in scores. There were significant increases in PA at T1 and T3 time-points with the EX group, and at T3 time-point only for MFI and CON, with MFI group showing largest increase in HRQOL scores. BMI increased significantly for all groups at T2 (MFI p=0.001, EX p<0.001) and T3 (p<0.001), while HRQOL increased significantly for both MFI and EX at both follow-ups (p<0.001).\n\nConclusions School-based lifestyle MFI was more effective for improving PA, lifestyle behaviours and HRQOL than exercise alone for adolescents, although BMI was not reduced. MFI with PA could be an effective school-based approach for behaviour modification but BMI has limitations for measuring body composition changes.\n\nRegistration\nCTRI/2019/04/018834 (30/04/2019).", "keywords": [ "Adolescents", "lifestyle", "diet", "physical activity", "parents", "health education", "school" ], "content": "1. Introduction\n\nAdolescent obesity is a global issue in developed as well as developing nations.1–3 A 2020 study predicted that by 2030, the number of obese and overweight adolescents will exceed 250 million globally, with China, India and USA being largest contributors.4 This has made weight gain a prevalent long term health issue among adolescents.4 Research shows there is 70%-80% likelihood of an overweight adolescent developing adulthood obesity, making them prone for short and long term health challenges.5,6 Therefore, effective weight management and prevention programs become crucial during adolescence.6\n\nInterventions targeting lifestyle modifications have been a significant focus and first step in clinical and public settings for addressing obesity among adolescents.5 In developing countries with resource-limited settings, lifestyle interventions offer a practical approach to address adolescent obesity.7 Such interventions emphasize the importance of reducing high calorie food intake, promoting healthy eating habits, minimizing sedentary behaviour, engaging in regular physical activity (PA) and implementing behavioural modifications to educate parents about sustaining a healthy lifestyle.8–12 Studies shows that making changes to the school food environment, such as banning sugary drinks and increasing availability of fruits and vegetables, led to a significant decrease in adolescent obesity rates.13,14 Research has backed strategies promoting nutritional education and focusing on healthy food behaviours to promote a well-rounded diet, aligning with dietary recommendations for adolescents.15–17 Furthermore, PA interventions for adolescents which included exercise promotion and sedentary time reduction in school settings have been found to be effective in lowering Body Mass Index (BMI).8,18,19 However, the literature indicates that interventions focusing on diet and PA individually have a lower impact compared to being used in combination.20 Furthermore, irrespective of body weight, increasing PA, reducing sedentary time and improving dietary patterns have been associated with reduced illness and better health outcomes, ultimately leading to an improved quality of life.21–23\n\nPrevious studies in India have primarily evaluated the effect of PA and dietary modifications separately using outcome measures such as anthropometry, food habits and changes in PA levels.24–26 These separate interventions have been moderately effective in the short term but have not provided long term solutions to the adolescent obesity problem. There is also limited literature on the role played by familial factors such as increasing family awareness of the importance of improving PA levels and healthy eating, which can significantly contribute to adolescent health.27 A study conducted by Nayak et al (2016) among Indian adolescents concluded that parental involvement in weight loss interventions through PA and dietary education, and long-term follow up, can be crucial.28 The Indian government also has recommended the need for implementing a systematic approach to improving health and promoting weight loss through primary and secondary prevention strategies.29 Further, these prevention strategies should be implemented through government programs, school-based PA interventions, and nutritional education initiatives.26,27\n\nThis cluster RCT aims to compare the effectiveness of a 12-week culturally appropriate, school-based PA and exercise intervention to an exercise-only intervention or no intervention for obese (OB) and overweight (OW) adolescents. We hypothesize that while both the interventions may increase PA, the lifestyle-based intervention will lead to more significant improvements in PA, BMI and health-related quality of life (HRQOL) and will be more effective in the long term.\n\n\n2. Methods\n\nThe study was conducted as per the principles of the Declaration of Helsinki and in accordance with the medical research involving human subject act (WMO). Permissions to approach the schools was sought from the Office of the Deputy Director of Public Instructions (DDPI), Udupi, Karnataka, India and each individual school principal’s permission was sought. The participants and their parents provided their informed consent at the beginning of the study and complete procedures were explained verbally and by administering the participant information sheet.\n\nThis study is a part of larger cluster randomized control trial, conducted for 12 weeks (T1),30 with follow-up assessments at6 (T2) and 12 months (T3) post-baseline. The study was based on the Theory of Planned Behaviour, which suggests that an individual’s perceptions can be changed by culturally sensitive knowledge, leading to an intention to change behaviour.31 The schools were randomized into three blocks based on geographical locations, with three schools in each block. A computer-generated randomization sequence was created for each block. The allocation concealment for the principal investigator was ensured by the placing of the random sequences in opaque envelopes by a faculty member of Manipal Academy who was not involved in the project. The adolescents from each school were divided into three groups – a multifactorial intervention (MFI group), an exercise-only intervention (EX group) and no intervention (CON group). Detailed procedures have been published previously.30\n\nThe inclusion criteria for the study were (1) males and females aged 11–16 years, who were attending English medium schools in Karnataka; (2) a BMI higher than the 85th percentile on the standard Centres for Disease Control BMI-for- age growth charts, where OW is at or above the 85th percentile and below the 95th percentile and OB is at or above the 95th percentile32; (3) Participants should pass health screening using Physical Activity Readiness for Everyone (PAR-Q+).33\n\nThe exclusion criteria were (1) medically-diagnosed obesity due underlying disease or medications; (2) undergoing psychiatric treatments; (3) inability to attend twice-weekly intervention sessions in schools; (4) declared unfit to participate by general practitioner or paediatrician; (5) participating in another structured exercise programme. If participants reported any mental issue such as anxiety or depression, related to study participation, they were offered counselling at school, local hospital or through social services.\n\nThe sample size was calculated based on an unpublished pilot study considering BMI Z-scores as a primary outcome measure. The level of significance (α) was adjusted for three comparisons and considered at 0.05/3 to be 2.59, with a power of 80%. The anticipated standard deviation of the outcome variable for the population would be 2.13 and minimum clinical difference was assumed to be 1.5. The intraclass correlation coefficient (ICC) was determined to be 0.3. The proposed total sample size was 600, with 200 adolescents in the three groups (MFI, EX, CON). The detailed study protocol has been published previously.30\n\nPrimary outcome measures\n\nThe level of (PA) of the adolescent participants was measured using the Physical Activity Questionnaire- Adolescent (PAQ-A),34 and BMI-Z scores using standard procedures.35\n\nSecondary outcome measure\n\nHealth-related quality of life (HRQOL) was assessed using Quality of Life Teen Report.\n\n(PedsQLTM 4.0).36 This questionnaire assesses physical function, emotional wellbeing, social functioning, and school performance.\n\nThe evaluations and interventions were conducted in schools by a physiotherapist. Baseline assessment was completed for all the adolescents regardless of randomized group. The MFI group received PA and dietary education, plus parental education on healthy lifestyles, at the beginning of the intervention. The education consisted of a validated education manual for adolescents and parents, developed by qualified healthcare professionals and based on standard guidelines,37 which reinforced culturally-adapted, age-specific healthy diet and optimum PA recommendations. The manual was printed in both English and the local language (Kannada) with illustrations of PA recommendations, dietary guidelines (e.g. portion size) and recommendations for decreasing electronic device screen time for adolescents. The education manual validation process has been explained in the published protocol.30\n\nThe MFI group was given exercises based on American College of Sports Medicine (ACSM) guidelines for 60 min, twice weekly during school physical education (PE) classes under the supervision of a physiotherapist and PE teacher. Each session included moderate to high-intensity aerobic and strengthening exercises, with guidance on intensity using the Pictorial Child Effort Rating Table (PCERT), based on heart rate and breathing. Aerobic exercises (e.g. running, jogging, brisk walking) and strengthening exercises (e.g. tug of war, body weight resistance exercises) were part of each session. In addition, PA and dietary educational sessions were held for the adolescents at the school, while similar sessions took place for parents during parent-teacher meetings at the beginning of the intervention. Post-intervention follow up was conducted at the school. Any adolescents missing sessions for two continuous weeks were considered for dropout.\n\nThe EX group were given exercise only during PE classes under the supervision of a physiotherapist and PE teacher, with no diet, PA and behavioural education. The CON adolescents were encouraged to continue their regular activities and diet, with no interventions. All three groups were reassessed (1) post-intervention block at T1 and (2) with follow-up contact at T2 and T3 post-intervention time points.\n\nStatistical analysis was performed using Jamovi software version 2.3.4. The demographic data was analysed using descriptive statistics (mean±SD), and Shapiro-Wilk tests were employed to assess the data normality. A general linear model with repeated measures and Bonferroni post hoc corrections at a level of significance of 0.05 was used to compare means across three groups and at different time points (group effect, time effect and group x time effect). Cohen’s effect size (ES) was used to indicate the magnitude of change with 0.2 being small, 0.5 medium and 1.2 being large.\n\n\n3. Results\n\nOut of a total of 3425 adolescents screened from nine schools, 671 obese (OB) and overweight (OW) adolescents participated in the study as per the inclusion criteria. These students were randomised into three groups with 224 in both the MFI and EX groups, and 223 in the CON group. After the follow-up period of 12 months, there were 197 adolescents in MFI group, 199 in EX group, and 208 in CON group due to dropouts. The CONSORT flowchart of the study is shown in Figure 1.\n\nThe mean age of 126 (64%) males and 71 (36%) females in MFI group was 12.7±1.3 years, compared to 13.1±1.2 years for 121 (61%) males and 78 (39%) females in the EX group and 13.1±1.4 years for 136 (65%) males, 72 (35%) females in the CON group. The baseline demographic details of participants are shown in Table 1.\n\nPhysical Activity\n\nAt T0, the mean PA scores were not significantly different (see Table 2). At T1, the score for the MFI group was 3.57±0.33, compared to the EX group with 3.50±0.31 and CON group with 3.43±0.35, At T3 the MFI group score was 4.55±0.32, while the EX and CON group changed to 3.91±0.33 and 3.60±0.33, respectively (Table 2).\n\nAnalysis of the time and group effects (Table 2) shows statistically significant changes in PA scores over all the time points within the study period (p<0.001), with a small to medium effect (ES=0.354). The overall group effect was also significant (p<0.001) with a small effect size (ES= 0.199). The group x time interaction was statistically significant (p<0.001) with a small effect size (ES=0.183). The post-hoc analysis shows that there were no significant post-intervention differences in PA scores between the three groups. However, there were significant differences between T0 and T1 follow up for the MFI group (p<0.001), T0 to T2 follow up and at T3 for the EX group (p<0.001) and between T0 and T3 for the CON group (p<0.001).\n\nBody Mass Index (BMI)\n\nAt T0, T1, T2 time-points, there were no significant differences between groups for mean BMI scores; (Table 3).\n\n* Significant difference p <0.05.\n\nAnalysis of the time and group effects (Table 3) shows statistically significant changes in BMI over all the time points (p<0.001) with a small effect size (ES= 0.237). The group effect was also significant (p<0.001) but with a negligible ES. The group x time interaction also shows statistically significant interaction (p<0.001) but with a negligible ES. The post-hoc analysis shows that the differences in BMI from T0 to T1 were non-significant for all groups (p=1.000). From T0 to T1, there were significant increases for the MFI (p = 0.001), EX (p<0.001) and CON (p<0.001) groups. From T0 to T3, there were again significant increases (p<0.001) for each group. The biggest BMI increase during the study was in the CON group, with the MFI group increasing their mean score the least.\n\nHealth Related Quality of life (HRQOL).\n\nIn mean HRQoL scores; at T3, the MFI group showed highest change, followed by EX group and CON group had the least increase in HRQOL scores. The HRQOL values are presented in Table 4. The results show a statistically significant main effect of time (p<0.001) with small ES of 0.266; group effects were also significant (p<0.001) with a small ES (0.311). The time and group interaction (p<0.001) also showed a small ES of 0.234. From T0 to T1, both MFI and EX groups increased their mean HRQOL scores significantly (p<0.001), compared to the CON group (p = 1.000). At T2 and T3, the MFI and EX groups also increased scores compared to baseline values (p<0.001 for both groups) while the CON did not.\n\n* Significant difference p<0.05.\n\n\n4. Discussion\n\nThe primary objective of this study was to evaluate how a 12-week with long term follow-up multifactorial lifestyle intervention impacted PA levels and BMI in overweight and obese adolescents, with a secondary focus on changes in HRQOL. The adolescents who received the MFI intervention showed greater improvements in self-reported PA and HRQOL compared to those in EX and CON groups. The BMI increased in all the three groups, however MFI group showed least change. Furthermore, these changes were largely sustained by the MFI and EX groups for up to one year after the intervention.\n\nThe positive effects observed in PA levels are extremely important in the context of improving adolescent health and reducing the risk of chronic disease onset.38,39 The changes could be credited to the supervised PA in the schools by PE teachers,40 the long-term follow ups,41 where teachers are in a unique position to motivate students and increase their self-awareness39,42 plus the individual tailoring of exercise intensity using the PCERT guide.43 Other factors that could contribute to the greater improvements in the MFI group include the educational and promotional component for students and families,44,45 and the implementation of a theory-based intervention in addition to the PA intervention.31 A review showed that school-based multifactorial interventions utilising PA promotional literature were linked positively to increased PA among adolescents.45 This further emphasizes the need for schools to provide enough opportunities for students to participate in regular PA. Previous studies in the USA46 and Australia47 have also demonstrated similar findings, where school-based integrative, multiple behaviour interventions were found to be effective in improving PA levels among adolescents. The authors attributed lifestyle modifications to increasing awareness of the benefits of PA, increasing fruits and vegetable consumption, and involving parents and teachers in the interventions for adolescents.\n\nWe also found a slight and unexpected PA increase at the 12-month follow-up in the CON group. It is possible that some CON adolescents provided PAQ-A responses they believed the principal investigator expected, thus scoring higher in the questionnaire than was accurate. This is one of the potential drawbacks of using self-reported PA rather than direct measures. Repeated administration of questionnaires at multiple time points may have prompted students to realize their inactive lifestyle and change it, which is a positive behavioural change in the context of PA. This change could be because of the Hawthorne effect48 or due to social desirability.49 The Hawthorne effect refers to the tendency to change one’s behaviour in response to being observed or given attention by the researcher.50,51 Several studies concluded that increases in PA among control group participants were attributed to social desirability, with participants providing responses that aligned with social expectations.52–54 The participant-investigator interaction effect can neither be validated nor discounted in the present study.\n\nIn terms of BMI, the present study showed increases in all the three groups but with the MFI group showing the least increase. This could be due to increases in regular PA which reduced body fat as well as increasing relative lean muscle mass in the MFI and EX groups, thus a change in body composition.39 As clinical indicators of weight gain, BMI classifications have limitations i.e. they do not take into account the proportions of fat mass and lean muscle mass components in total weight mass.55 Therefore, BMI cut-off values may have high specificity in obesity diagnoses but low sensitivity to identify adiposity.56\n\nThe multifaceted lifestyle approach of the intervention may also have contributed to positive BMI results for the MFI group. The intervention included parental support, dietary education on healthy eating with instructions and benefits of consuming higher quantities of vegetables and fruits while cutting down on calorie-dense food with culturally-appropriate illustrations. Also, the logbook embedded in the educational booklet plus regular participant follow-ups may have contributed to higher adherence to a healthy lifestyle among the MFI group.57 These result align with a goal of the study to reduce negative energy imbalance among adolescents by encouraging better food choices and higher PA levels.\n\nThe perceived higher HRQOL in the MFI group compared to the EX and CON groups may be due to a variety of factors associated with the intervention, especially increased education, parental input and behaviour changes. HRQOL is influenced by sociodemographic and psychological factors, lifestyle choices, physical health, and family relationships.58–60 Research indicates that children often adopt their parent’s lifestyle patterns, so it’s important for the parents to be educated and motivated to make healthy changes such as promoting healthy eating patterns, reducing screen time, demonstrating positive family dynamics through parental affection and autonomy promotion.58–60 Along with parental education, regular PA involvement can have positive impact on various psychosocial factors like self-esteem, self-image, self-efficacy, social and peer interactions, and school performance.58–60 Regular PA has also been associated with improved physical health parameters and reduced risk of chronic diseases.61 These factors may directly or indirectly contribute towards better HRQOL among adolescents. However, our HRQOL findings are in contrast with studies by Resaland et al and Raj et al, where no change in HRQOL was observed among school-going participants.61,62 The lack of change in these studies was concluded to be due to the interventions focusing only on PA for a short duration62 and that the study population was treatment-seeking rather than being randomly allocated.58 In our study, the long-term follow-ups assessing HRQOL and PA levels were extremely important to determine the sustainability of the multifactorial intervention benefits physically and psychologically.\n\nThe study has strengths like cluster RCT design and long-term follow-ups for lifestyle changes. However, limitations include English medium school population sample, therefore results should be applied to government schools with caution, non-blinding of outcome measures and subjective assessment of PA due lack of funding and resources. Subjective measures like questionnaires can lead to over or under estimation of PA. To combat obesity, integrating lifestyle education, PA guidelines, and health promotion into school curriculums should be considered at policy-making levels. Evaluating policy-based interventions like replacing high-calorie food with healthier options should also be evaluated.\n\n\nConclusion\n\nThe study suggests that a multifactorial lifestyle intervention, including education and regular exercise, is more effective in increasing physical activity levels and preventing BMI increases in overweight adolescents. Future studies with objective physical activity assessments into PA and lifestyle interventions are recommended.\n\n\nEthics and consent\n\nThe study was conducted as per the principles of the Declaration of Helsinki and in accordance with the medical research involving human subject act (WMO). The current study is part of larger ongoing randomized trial. The study was approved by IRC-Institutional Research Committee (23/06/2018) and IEC- Institutional Ethics Committee of Kasturba Medical College and Kasturba Hospital Manipal (19/09/2018) (IEC: 536-2018). The clinical trial was registered in Clinical Trials Registry - India, Trial number CTRI/2019/04/018834, registered on 30/04/2019 (https://www.ctri.nic.in/Clinicaltrials/pmaindet2.php?EncHid=MzE1Mzk=&Enc=&userName=CTRI/2019/04/018834). Permissions to approach the schools was sought from the Office of the Deputy Director of Public Instructions (DDPI), Udupi, Karnataka, India and each individual school principal’s permission was sought.\n\nEach participant and their parents signed a written informed consent before participation. Participants received study objective, procedures and rights to withdraw any time during these study through participant information sheet. The study participation was entirely voluntary.", "appendix": "Data availability\n\nFigshare: Adolescent PA, BMI and HRQOL. https://doi.org/10.6084/m9.figshare.26123824. 64\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: CONSORT checklist for ‘Effect of a pragmatic lifestyle modification intervention on physical activity levels and body mass index among obese and overweight adolescents in Udupi, India: A cluster randomized trial’. https://doi.org/10.6084/m9.figshare.26124562. 63\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nHarvard Dataverse: Replication Data for: Fig 1 Consort flowchart of the study. https://doi.org/10.7910/DVN/ADFWTX. 65\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nThe authors would like to acknowledge all the participants and their parents for voluntarily participating in the study. We would also like to acknowledge all the school principals for allowing us to enrol their schools in the study. We would like to thank all the questionnaire authors for permitting to their questionnaires free of cost for this non-funded study.\n\n\nReferences\n\nKansra AR, Lakkunarajah S, Jay MS: Childhood and Adolescent Obesity: A Review. Front. Pediatr. 2021; 8(581461). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbarca-Gómez L, Abdeen ZA, Hamid ZA, et al.: Worldwide trends in body-mass index, underweight, overweight, and obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement studies in 128·9 million children, adolescents, and adults. Lancet. 2017; 390(10113): 2627–2642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLister NB, Baur LA, Felix JF, et al.: Child and adolescent obesity. Nat. Rev. Dis. Primers. 2023; 9(1): 19–24. PubMed Abstract \| Publisher Full Text\n\nLuhar S, Timæus IM, Jones R, et al.: Forecasting the prevalence of overweight and obesity in India to 2040. PLoS ONE. 2020; 15(2): e0229438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCardel MI, Atkinson MA, Taveras EM, et al.: Obesity Treatment Among Adolescents. JAMA Pediatr. 2020; 174(6): 609–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNicolucci A, Maffeis C: The adolescent with obesity: what perspectives for treatment?. Ital. J. Pediatr. 2022; 48(1): 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaillot A, Romain AJ, Boisvert-Vigneault K, et al.: Effects of Lifestyle Interventions That Include a Physical Activity Component in Class II and III Obese Individuals: A Systematic Review and Meta-Analysis. Kaser S, ed. PLOS ONE. 2015; 10(4): e0119017. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMei H, Xiong Y, Xie S, et al.: The impact of long-term school-based physical activity interventions on body mass index of primary school children – a meta-analysis of randomized controlled trials. BMC Public Health. 2016; 16(1): 205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViitasalo A, Eloranta AM, Lintu N, et al.: The effects of a 2-year individualized and family-based lifestyle intervention on physical activity, sedentary behavior and diet in children. Prev. Med. 2016; 87: 81–88. PubMed Abstract \| Publisher Full Text\n\nGraf C, Rost SV, Koch B, et al.: Data from the StEP TWO programme showing the effect on blood pressure and different parameters for obesity in overweight and obese primary school children. Cardiol. Young. 2005; 15(3): 291–298. PubMed Abstract \| Publisher Full Text\n\nHofsteenge GH, Weijs PJ, Delemarre-van de Waal HA, et al.: Effect of the Go4it multidisciplinary group treatment for obese adolescents on health related quality of life: a randomised controlled trial. BMC Public Health. 2013; 13(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchranz N, Tomkinson G, Parletta N, et al.: Can resistance training change the strength, body composition and self-concept of overweight and obese adolescent males? A randomised controlled trial. Br. J. Sports Med. 2013; 48(20): 1482–1488. PubMed Abstract \| Publisher Full Text\n\nFlieh SM, Moreno LA, Miguel-Berges ML, et al.: Free Sugar Consumption and Obesity in European Adolescents: The HELENA Study. Nutrients. 2020; 12(12): 3747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGandhi AB: Diet and Weight Management in Adolescent Girls. The Journal of Obstetrics and Gynecology of India. 2022; 72(2): 175–177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGato-Moreno M, Martos-Lirio MF, Leiva-Gea I, et al.: Early nutritional education in the prevention of childhood obesity. Int. J. Environ. Res. Public Health. 2021; 18(12): 6569. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardiner CK, Bryan AD: Monetary Incentive Interventions Can Enhance Psychological Factors Related to Fruit and Vegetable Consumption. Ann. Behav. Med. 2017; 51(4): 599–609. PubMed Abstract \| Publisher Full Text\n\nJurić P, Jurak G, Morrison SA, et al.: Effectiveness of a population-scaled, school-based physical activity intervention for the prevention of childhood obesity. Obesity. 2023; 31(3): 811–822. PubMed Abstract \| Publisher Full Text\n\nSacchetti R, Ceciliani A, Garulli A, et al.: Effects of a 2-Year School-Based Intervention of Enhanced Physical Education in the Primary School. J. Sch. Health. 2013; 83(9): 639–646. PubMed Abstract \| Publisher Full Text\n\nHarris KC, Kuramoto LK, Schulzer M, et al.: Effect of school-based physical activity interventions on body mass index in children: a meta-analysis. Can. Med. Assoc. J. 2009; 180(7): 719–726. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersen E, van der Ploeg HP , van Mechelen W , et al.: Contributions of changes in physical activity, sedentary time, diet and body weight to changes in cardiometabolic risk. Int. J. Behav. Nutr. Phys. Act. 2021; 18(1): 166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRendo-Urteaga T, Augusto, Collese TS, et al.: The combined effect of physical activity and sedentary behaviors on a clustered cardio-metabolic risk score: The Helena study. Int. J. Cardiol. 2015; 186(1): 186–195. PubMed Abstract \| Publisher Full Text\n\nShi L, Morrison JA, Wiecha J, et al.: Healthy lifestyle factors associated with reduced cardiometabolic risk. Br. J. Nutr. 2011; 105(5): 747–754. Publisher Full Text\n\nCarducci B, Oh C, Keats EC, et al.: Effect of Food Environment Interventions on Anthropometric Outcomes in School-Aged Children and Adolescents in Low- and Middle-Income Countries: A Systematic Review and Meta-Analysis. Current Developments in Nutrition. 2020; 4(7): nzaa098. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJalambo MO, Karim NA, Naser IA, et al.: Prevalence and risk factor analysis of iron deficiency and iron-deficiency anaemia among female adolescents in the Gaza Strip, Palestine. Public Health Nutr. 2018; 21(15): 2793–2802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh AK, Maheshwari A, Sharma N, et al.: Lifestyle associated risk factors in adolescents. The Indian Journal of Pediatrics. 2006; 73(10): 901–906. Publisher Full Text\n\nKalpana CA, Lakshmi UK, Devi SR: Impact of different intervention strategies on selected obese children of Coimbatore City. Indian J Nutr Diet. 2010; 47(5): 179–187.\n\nBrown SL, Manning WD, Stykes JB: Family Structure and Child Well-being: Integrating Family Complexity. J. Marriage Fam. 2015; 77(1): 177–190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNayak BS, Bhat VH: School Based Multicomponent Intervention for Obese Children in Udupi District, South India – a Randomized Controlled Trial. J. Clin. Diagn. Res. 2016; 10(12): SC24–SC28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRVP S, Aayog N: Minutes of the third meeting of the National technical board on nutrition 12th April 2019. A scientific consultation on childhood and adolescent overweight-obesity in India., 2019. [Accessed 16 April 2024]. Reference Source\n\nSrivastav P, Vaishali K, Bhat VH, et al.: Structured, multifactorial randomised controlled intervention to investigate physical activity levels, body composition and diet in obese and overweight adolescents. BMJ Open. 2021; 11(3): e044895. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBosnjak M, Ajzen I, Schmidt P: The Theory of Planned behavior: Selected Recent Advances and Applications. Eur. J. Psychol. 2020; 16(3): 352–356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe M, Sutton J: Using Routine Growth Monitoring Data in Tracking Overweight Prevalence in Young Children. Can. J. Public Health. 2004; 95(6): 419–423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarburton D, Jamnik VK, Bredin S, et al.: The Physical Activity Readiness Questionnaire for Everyone (PAR-Q+) and Electronic Physical Activity Readiness Medical Examination (ePARmed-X+). The Health & Fitness Journal of Canada. 2011; 4(2): 3–17. Publisher Full Text\n\nVoss C, Dean PH, Gardner RF, et al.: Validity and reliability of the Physical Activity Questionnaire for Children (PAQ-C) and Adolescents (PAQ-A) in individuals with congenital heart disease. Buchowski M, ed. PLOS ONE. 2017; 12(4): e0175806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInokuchi M, Matsuo N, Takayama JI, et al.: BMI z-score is the optimal measure of annual adiposity change in elementary school children. Ann. Hum. Biol. 2011; 38(6): 747–751. PubMed Abstract \| Publisher Full Text\n\nVarni JW, Seid M, Kurtin PS: PedsQLTM 4.0: Reliability and Validity of the Pediatric Quality of Life InventoryTM Version 4.0 Generic Core Scales in Healthy and Patient Populations. Med. Care. 2001; 39(8): 800–812. PubMed Abstract \| Publisher Full Text\n\nLee H, Kim J, Yoo R, et al.: Development and Evaluation of Cardiovascular Disease Prevention Education Materials for Middle-aged Korean-Chinese Female Workers: Applying Patient Education Materials Assessment Tool for Printable Materials (PEMAT-P). J. Korean Acad. Community Health Nurs. 2016; 27(3): 284. Publisher Full Text\n\nRemmert JE, Woodworth A, Chau L, et al.: Pilot Trial of an Acceptance-Based Behavioral Intervention to Promote Physical Activity Among Adolescents. J. Sch. Nurs. 2018; 35(6): 449–461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaegele JA, Wilson WJ, Zhu X, et al.: Barriers and facilitators to inclusion in integrated physical education: Adapted physical educators’ perspectives. Eur. Phys. Educ. Rev. 2020; 27(2): 297–311. Publisher Full Text\n\nMak TCT, Chan DKC, Capio CM: Strategies for Teachers to Promote Physical Activity in Early Childhood Education Settings—A Scoping Review. Int. J. Environ. Res. Public Health. 2021; 18(3): 867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCassar S, Salmon J, Timperio A, et al.: Adoption, implementation and sustainability of school-based physical activity and sedentary behaviour interventions in real-world settings: a systematic review. Int. J. Behav. Nutr. Phys. Act. 2019; 16(1): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKasai D, Parfitt G, Tarca B, et al.: The Use of Ratings of Perceived Exertion in Children and Adolescents: A Scoping Review. Sports Med. 2020; 51(1): 33–50. PubMed Abstract \| Publisher Full Text\n\nHa AS, Ng JYY, Lonsdale C, et al.: Promoting physical activity in children through family-based intervention: protocol of the “Active 1 + FUN” randomized controlled trial. BMC Public Health. 2019; 19(1): 218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbert FA, Crowe MJ, Malau-Aduli AEO, et al.: Physical Activity Promotion: A Systematic Review of The Perceptions of Healthcare Professionals. Int. J. Environ. Res. Public Health. 2020; 17(12): 4358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWerch CE, Bian H, Carlson JM, et al.: Brief integrative multiple behavior intervention effects and mediators for adolescents. J. Behav. Med. 2010; 34(1): 3–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSutherland RL, Campbell EM, Lubans DR, et al.: The Physical Activity 4 Everyone Cluster Randomized Trial. Am. J. Prev. Med. 2016; 51(2): 195–205. PubMed Abstract \| Publisher Full Text\n\nMcCambridge J, Witton J, Elbourne DR: Systematic Review of the Hawthorne effect: New Concepts Are Needed to Study Research Participation Effects. J. Clin. Epidemiol. 2014; 67(3): 267–277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJúnior B, Patrício J: Social desirability bias in qualitative health research. Rev. Saude Publica. 2022; 56(56): 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCepoiu M, McCusker J, Cole MG, et al.: Recognition of Depression in Older Medical Inpatients. J. Gen. Intern. Med. 2007; 22(5): 559–564. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErtem IO, Votto N, Leventhal JM: The Timing and Predictors of the Early Termination of Breastfeeding. Pediatrics. 2001; 107(3): 543–548. PubMed Abstract \| Publisher Full Text\n\nCovelli MM: Efficacy of a school-based cardiac health promotion intervention program for African-American adolescents. Appl. Nurs. Res. 2008; 21(4): 173–180. PubMed Abstract \| Publisher Full Text\n\nGnambs T, Kaspar K: Socially Desirable Responding in Web-Based Questionnaires: A Meta-Analytic Review of the Candor Hypothesis. Assessment. 2016; 24(6): 746–762. PubMed Abstract \| Publisher Full Text\n\nBergen N, Labonté R: “Everything Is perfect, and We Have No problems”: Detecting and Limiting Social Desirability Bias in Qualitative Research. Qual. Health Res. 2020; 30(5): 783–792. PubMed Abstract \| Publisher Full Text\n\nMoon JS, Lee SY, Nam CM, et al.: 2007 Korean National Growth Charts: review of developmental process and an outlook. Clinical and Experimental Pediatrics. 2008; 51(1): 1–25. Publisher Full Text\n\nOkorodudu DO, Jumean MF, Montori VM, et al.: Diagnostic performance of body mass index to identify obesity as defined by body adiposity: a systematic review and meta-analysis. International journal of obesity (2005). 2010; 34(5): 791–799. PubMed Abstract \| Publisher Full Text\n\nCollado-Mateo D, Lavín-Pérez AM, Peñacoba C, et al.: Key Factors Associated with Adherence to Physical Exercise in Patients with Chronic Diseases and Older Adults: An Umbrella Review. Int. J. Environ. Res. Public Health. 2021; 18(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikkelsen HT, Småstuen MC, Haraldstad K, et al.: Changes in health-related quality of life in adolescents and the impact of gender and selected variables: a two-year longitudinal study. Health Qual. Life Outcomes. 2022; 20(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZashikhina A, Hagglof B: Health-related quality of life in adolescents with chronic physical illness in northern Russia: a cross-sectional study. Health Qual. Life Outcomes. 2014; 12(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiménez-Iglesias A, Moreno C, Ramos P, et al.: What family dimensions are important for health-related quality of life in adolescence?. J. Youth Stud. 2014; 18(1): 53–67. Publisher Full Text\n\nGualdi-Russo E, Zaccagni L: Physical Activity for Health and Wellness. Int. J. Environ. Res. Public Health. 2021; 18(15). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResaland GK, Aadland E, Moe VF, et al.: Effects of a physical activity intervention on schoolchildren’s health-related quality of life: The active smarter kids (ASK) cluster-randomized controlled trial. Prev. Med. Rep. 2019; 13(3): 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaj M, Sudhakar A, Roy R, et al.: Health-related quality of life (HRQOL) in children and adolescents with congenital heart disease: a cross-sectional survey from South India. BMJ Paediatrics Open. 2019; 3(1): e000377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSrivastav P: CONSORT Checklist Lifestyle manuscript.docx. [Dataset]. figshare. 2024. Publisher Full Text\n\nSrivastav P: Adolescent PA, BMI and HRQOL. [Dataset]. figshare. 2024. Publisher Full Text\n\nSrivastav P, Vaishali K, Vinod Bhat H, et al.: Replication Data for: Fig 1 Consort flowchart of the study Harvard Dataverse, V1.2024. Publisher Full Text" }	[ { "id": "310425", "date": "05 Aug 2024", "name": "Snehal Subrat Samal", "expertise": [ "Reviewer Expertise Neurophysiothearpy", "Musculoskeletal Physiotherapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 thank the journal for providing me with the opportunity to review the article. The article articulates very nicely that it is the need of the hour for adolescent obesity intervention in India. There are a few suggestions for making the study better:\n1) The study mentions the importance of parental involvement in lifestyle modifications. Still, providing more detailed information on how parents were engaged and the specific activities they participated in would be beneficial.\n2) While the study includes long-term follow-ups, more details on the frequency and nature of these follow-ups would help understand their impact on the sustainability of the intervention benefits.\n3) The educational and promotional components for students and families are mentioned as contributing factors to the success of the intervention. Providing more specifics about the content and delivery methods of these educational materials would enhance the understanding of their effectiveness\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "310428", "date": "06 Aug 2024", "name": "Jaya Shanker Tedla", "expertise": [ "Reviewer Expertise Physical Therapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs a reviewer of this paper, I appreciate the multifactorial approach adopted by the authors to see the effects on school children. The effect sizes reported in this study can guide future research by providing benchmarks for expected changes. Researchers can use these benchmarks to design studies with adequate power to detect meaningful effects. However, there are few clarifications required.\n1) The intervention is described as culturally appropriate. Elaborating on how it was tailored to fit the participants' cultural context would provide valuable insights for replicating the study in other settings.\n2) A more detailed comparison with similar studies conducted in different countries or regions could benefit the discussion. This would help contextualize the findings and highlight the unique aspects of the intervention.\n3) Suggesting specific areas for future research, such as exploring the long-term impact of the intervention on different age groups or in different settings, would be beneficial for advancing the field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/13-859
https://f1000research.com/articles/13-679/v1	24 Jun 24	{ "type": "Genome Note", "title": "The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae)", "authors": [ "Gert Wörheide", "Warren R. Francis", "Fabian Deister", "Stefan Krebs", "Dirk Erpenbeck", "Sergio Vargas", "Warren R. Francis", "Fabian Deister", "Stefan Krebs", "Dirk Erpenbeck", "Sergio Vargas" ], "abstract": "Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.", "keywords": [ "Tethya wilhelma", "Tethya minuta", "Porifera", "Demospongiae", "genome", "model organism" ], "content": "Introduction\n\nSponges (Phylum Porifera) are sessile aquatic metazoans that occur globally in marine and freshwater habitats. More than 9,600 valid species have been described, the majority of which (7,989 species) belong to Class Demospongiae.1 Sponges hold a pivotal position in the animal tree of life as one of the earliest metazoan branching lineages, likely originating more than 650 million years ago,2 but their exact phylogenetic position is still disputed.3–6 As filter feeders, sponges are ecologically important, especially in coral reefs,7,8 but are also impacted by climate change, as they bleach due to elevated seawater temperatures.9,10 In the current biodiversity crisis, genome data is valuable to aid species conservation11 and genomic data can be used not only to understand evolution and development,12 but also to predict a species evolutionary potential to adapt to changing environmental conditions due to climate change.13 To improve the understanding of their response to changing environmental conditions, the availability of culturable sponge species with annotated high-quality genomes is important, but only a few sponge species meet both these criteria yet.\n\nAlthough the first sponge genome was published in 2010 from the Australian demosponge Amphimedon queenslandica,14 only a handful of annotated high-quality sponge genomes have been published and analysed since then, for example from the freshwater demosponge Ephydatia muelleri15 or the reef-building glass sponge (Class Hexactinellida) Aphrocallistes vastus.16 However, only A. queenslandica and E. muelleri are culturable yet under controlled conditions. This lack of high-quality genome resources available from culturable sponges hinders a full appreciation of our understanding of animal evolution and function as well as the response of sponges as ecological key players in many aquatic ecosystems to the current climate crisis.\n\nTo contribute to filling this gap, we here provide high-quality draft genomes of the two aquarium species Tethya wilhelma and Tethya minuta. These two congeners are small ball-shaped demosponges that were described in 2001 from public aquaria in Germany.17 Due to their long-term culturability, they are a laboratory model for many topics, including multicellularity, early-animal evolution, biomineralization, and even cancer (e.g., Refs. 18–25). With the provision of novel high-quality genome data of these two species we aim to enhance the use of these species as valuable sponge model systems.\n\n\nMethods\n\nSpecimens of Tethya wilhelma and Tethya minuta were obtained from the marine research aquaria of the Chair of Paleontology and Geobiology of the Department of Earth and Environmental Sciences at the Ludwig-Maximilians-Universität München (Germany), where they are cultured since about 2010. No permits were needed for the sampling and processing. Voucher specimens are deposited in the Bavarian State Collection for Paleontology and Geology (SNSB-BSPG) under accession numbers SNSB-BSPG.GW33333 (T. wilhelma) and SNSB-BSPG.GW41624 (T. minuta).\n\nFor both species, genomic DNA was extracted from either fresh or frozen tissue with a modified cetyltrimethylammonium bromide (CTAB; Carl Roth, Germany, Cat. Nr. 9161.1) extraction.26 Short DNA fragments were removed using AMPure XP (Beckman Coulter, USA, Cat. Nr. A63881) beads to select for long DNA fragments. DNA quantity and quality were controlled on a Nanodrop 1100 and using 1.5% agarose (Biozym, Germany, Cat. Nr. 840004) gels before library preparation as required for the different sequencing platforms used.\n\nFor Tethya wilhelma, we took the genome draft (T. wilhelma-v1) published by our group in 201720 as a starting point and used new sequence data and bioinformatics to further improve it. Additional data was obtained using Hi-C27 and Chicago (Dovetail Genomics28) libraries. For this, four whole sponges were frozen in liquid nitrogen and shipped to Dovetail Genomics (Scotts Valley, CA) for library preparation and sequencing. The resulting Chicago/Hi-C reads were processed using Dovetail’s proprietary software HiRise.28 After Chicago/Hi-C scaffolding, the assembly (dubbed T. wilhelma-v2) had 1,353 scaffolds, totaling 139 Mb, with an N50 of 5.5 Mb.\n\nWhile some chromosome-sized scaffolds were evident in T. wilhelma’s-v2 assembly, many putative chromosomes remained fragmented. Therefore, we tried to improve the assembly’s contiguity by adding Moleculo long reads as well as Nanopore long reads, the latter derived from a single run of an Oxford Nanopore MinION (see Table 1). The data was assembled using the programs “SSPACE_Long_Read v1-1”29 and “GapCloser v.1.12”.30 This assembly version, called Twi-v3, contained 967 scaffolds, totaling 138.92 Mb, with an N50 of 6.1 Mb (see Table 2).\n\nFor the assembly of T. wilhelma’s genome v4, high molecular weight DNA was extracted, and quality was assessed using a Nanodrop 1100. Fragment size was controlled on a 1.5 agarose gel and an Agilent 2200 TapeStation. Libraries for 10X Genomics (Pleasanton, CA) were generated and sequenced at the University of Potsdam, in collaboration with the group of Prof. M. Hofreiter (Evolutionary Adaptive Genomics, University of Potsdam, Germany), on an Illumina Nextseq500. About 390 M reads were obtained (Table 1) and assembled using the 10X-Genomics software “Supernova 2.1.1”.31 These assembled contigs were then used for scaffolding the T. wilhelma v3 assembly using “SSPACE-LongRead v1-1”29 and then with “P_RNA_scaffolder”32 using 100.7 M PE (125 bp) and 237 M RNA reads (25.2 Gb). Finally, we used hicstuff 2.3.033 and the Hi-C data available to create a contact map of the T. wilhelma assembly. This assembly, called Twi-v4, had 891 scaffolds with a total size of 138.9 Mb and an N50 of 6.7 Mb, which also included bacterial scaffolds (see Table 2 and below).\n\nFor the assembly of Tethya minuta, DNA of a single specimen of Tethya minuta (sample# GW41624) was extracted with CTAB26 and sequenced twice, using Oxford Nanopore PromethION (12.73 Gb of long reads) and MinION34 (2.17 Gb of long reads). Additionally, we Illumina-sequenced 27 Mbp paired-end (100 PE and 150 PE). These data were assembled with wtdbg2,35 and polished using minimap2.36 SSPACE_LongRead29 was used with the available nanopore data to scaffold the assembly. Finally, we used GapCloser 1.1230 and the available PE reads to close gaps in the assembly which then had a length of 139 Mb and consisted of 1,043 scaffolds (Tmi-v4), but still included bacterial contigs (see Table 3).\n\nFrom earlier versions of the genome, it was clear that Tethya wilhelma harbours two associated bacteria, both alphaproteobacteria, with an unknown interaction. With the relatively large scaffolds in the assembly, a clear split was seen in GC content and read mapping coverage (Figure 1). Consequently, we separated all scaffolds with GC content under 47% and defined those as sponge. The remaining scaffolds for the two bacteria were binned using “MetaBAT v2.15-25”,37 with default parameters. For T. wilhelma, this yielded 6 bins (see public data repository at Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/03-bacteria) with 1 bin corresponding to a single Rhizobiales species (genome appx 7.5 Mb), and the other 5 bins corresponding to a Roseobacter species (genome appx 4.8 Mb). These scaffolds were removed from the final Tethya wilhelma genome version. This assembly, called Twi-v4-no_bacteria, had 557 scaffolds with a total size of 126.1 Mb and an N50 of 6.7 Mb (Table 2; see Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/06-FINAL_Assemblies ENA accession GCA_964030475).\n\nMetaBAT v2.15-25 was used for binning. Unidentified scaffolds are shown in grey.\n\nFor T. minuta, we used “MetaBAT v2.15-25”37 to identify and separate bacterial contigs from sponge scaffolds, which had produced 23 bins from the assembly. One of the bins, numbered as bin-17, contained the bulk of the assembly, and was identified as originating from the sponge due to the GC content of 38.3% and substantial RNAseq mapping. This bin (here now called Tmi-v4-no_bacteria, Table 3) had 244 scaffolds with a total size of 86.07 Mb, around 40 Mb smaller than the assembly of T. wilhelma (Table 2, see Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/06-FINAL_Assemblies; ENA accession GCA_964030485). Nearly all of the large chromosomal pieces in T. wilhelma had matching pieces among the scaffolds of T. minuta, as evident on the synteny plot (Figure 2), which suggested that the assembly of T. minuta was smaller not because of missing scaffolds or mis-assemblies, but merely from a smaller genome.\n\nRNA was extracted from fresh tissue of Tethya minuta using TRIzol (Fisher Scientific, Germany, Cat. Nr. 12034977) and chloroform (Carl Roth, Germany, Cat. Nr. 3313.1) precipitation39 with subsequent quality control on a Bioanalyzer 2100. Libraries were prepared and sequenced twice using one third of a lane of an Illumina HiSeq1500 (100 bp and 50 bp) at the LMU GeneCenter, yielding 137 M read pairs. Reads were assembled de novo using Trinity,40 using default parameters, resulting in an assembly of 151,079 contigs with an average length of 677 bp. This assembly was also used as a training set for de novo gene prediction (see below). Transcriptome sequencing and assembly of Tethya wilhelma has been described in Francis et al.20 Statistics of the different sequencing libraries of Tethya wilhelma and Tethya minuta are given in Table 1.\n\nBoth Tethya species were annotated using AUGUSTUS. For T. wilhelma, we used the BRAKER v2.0 pipeline,41 with the options --useexisting --species=Tethya_wilh and including mapped RNA. This predicted a total of 28,113 gene models, which were used for downstream analysis.\n\nFor Tethya minuta, the assembly Tmi-v4-no_bacteria and the de novo Trinity assembly were used as inputs for WebAUGUSTUS.42 This yielded 22,779 gene models and 33,041 genes (see files ‘hints_pred’ and ‘hints_UTR_pred’ at Ref. 38 or https://github.com/PalMuc/2Tethya_genomes/tree/main/05-annotation/tethya_minuta_augustus).\n\n\nResults\n\nThe final version of the Tethya wilhelma draft genome assembly (see Table 2, Twi-v4-no_bacteria) without bacterial scaffolds has 557 scaffolds, a length of 126.1 MB, an N50 of 6.7 MB, and contains 1030.7 kb gaps (Ns). The final version of the Tethya minuta draft genome assembly (see Table 3, Tmi-v4-no_bacteria) without bacterial scaffolds has 244 scaffolds, a length of 86.07 MB, an N50 of 969.3 kb, and contains 534.5 kb gaps (Ns). BUSCO values for the two assemblies are given in Table 4.\n\nFor work with sponges (Porifera) no ethical clearing is needed.", "appendix": "Data availability\n\nRaw reads are available from the European Nucleotide Archive under bioproject numbers PRJNA288690, PRJEB53671, for individual accession numbers see Table 1.\n\nThe assembled genomes are also available in the European Nucleotide Archive: Tethya wilhelma GCA_964030475, Tethya minuta GCA_964030485.\n\nFurther data on the genome assemblies, including analytical pipelines and scripts, is available in the public repository https://github.com/PalMuc/2Tethya_genomes, archived at Zenodo (https://zenodo.org/doi/10.5281/zenodo.10991740). 38\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY-SA 4.0 DEED) (https://creativecommons.org/licenses/by-sa/4.0/).\n\nVoucher specimens are deposited in the Bavarian State Collection for Paleontology and Geology (SNSB-BSPG, https://bspg.snsb.de) under accession numbers SNSB-BSPG.GW33333 (T. wilhelma) and SNSB-BSPG.GW41624 (T. minuta).\n\n\nAcknowledgements\n\nM. Hofreiter and M. Preick (Evolutionary Adaptive Genomics, University of Potsdam) are acknowledged for assistance with 10X Genomics sequencing, as well as Helmut Blum (Laboratory for Functional Genome Analysis, LAFUGA, Gene Center, LMU München) for Oxford Nanopore PromethION and Illumina sequencing. Gabriele Büttner and Simone Schätzle are thanked for their valuable assistance in the laboratory work. We would like to thank M. Eitel for contributing to the sponge genome assemblies and for helpful discussions and comments. GW dedicates this work to his late wife Connie Wörheide, who left us too early during the preparation of this manuscript.\n\n\nReferences\n\nde Voogd NJ , Alvarez B, Boury-Esnault N, et al.: World Porifera Database. World Porifera Database; 2024 [cited 2024 Mar 20]. Reference Source\n\nDohrmann M, Wörheide G: Dating early animal evolution using phylogenomic data. Sci. Rep. 2017 Jun 15; 7(1): 3599. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuravel K, Porras L, Höhna S, et al.: Exploring genome gene content and morphological analysis to test recalcitrant nodes in the animal phylogeny. PLoS One. 2023 Mar 23; 18(3): e0282444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchultz DT, Haddock SHD, Bredeson JV, et al.: Ancient gene linkages support ctenophores as sister to other animals. Nature. 2023 May 17; 618: 110–117. Publisher Full Text\n\nTelford MJ, Moroz LL, Halanych KM: Evolution: A sisterly dispute. Nature. 2016 Jan 21; 529(7586): 286–287. PubMed Abstract \| Publisher Full Text\n\nRedmond AK, McLysaght A: Evidence for sponges as sister to all other animals from partitioned phylogenomics with mixture models and recoding. Nat. Commun. 2021 Mar 19; 12(1): 1–14. Publisher Full Text\n\nDe Goeij JM, van Oevelen D , Vermeij MJA, et al.: Surviving in a aarine desert: The Sponge Loop retains resources within coral reefs. Science. 2013 Oct 3; 342(6154): 108–110. PubMed Abstract \| Publisher Full Text\n\nOlinger LK, McClenaghan B, Hajibabaei M, et al.: Looking for the sponge loop: analyses of detritus on a Caribbean forereef using stable isotope and eDNA metabarcoding techniques. PeerJ. 2024 Feb 23; 12: e16970. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBell JJ, Smith RO, Micaroni V, et al.: Marine heat waves drive bleaching and necrosis of temperate sponges. Curr. Biol. 2023 Jan 9; 33(1): 158–163.e2. PubMed Abstract \| Publisher Full Text\n\nPerkins NR, Monk J, Soler G, et al.: Bleaching in sponges on temperate mesophotic reefs observed following marine heatwave events. Clim. Change Ecol. 2022 Dec 1; 3: 100046. Publisher Full Text\n\nTheissinger K, Fernandes C, Formenti G, et al.: How genomics can help biodiversity conservation. Trends Genet. 2023 Jul; 39(7): 545–559. Publisher Full Text\n\nRenard E, Leys SP, Wörheide G, et al.: Understanding animal evolution: The added value of sponge transcriptomics and genomics: The disconnect between gene content and body plan evolution. Bioessays. 2018 Sep; 40(9): e1700237. PubMed Abstract \| Publisher Full Text\n\nWaldvogel AM, Feldmeyer B, Rolshausen G, et al.: Evolutionary genomics can improve prediction of species’ responses to climate change. Evol. Lett. 2020 Feb; 4(1): 4–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSrivastava M, Simakov O, Chapman J, et al.: The Amphimedon queenslandica genome and the evolution of animal complexity. Nature. 2010 Jan 1; 466(7307): 720–726. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenny NJ, Francis WR, Rivera-Vicéns RE, et al.: Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri. Nat. Commun. 2020 Jul 27; 11(1): 1–11. Publisher Full Text\n\nFrancis WR, Eitel M, Vargas S, et al.: The genome of the reef-building glass sponge Aphrocallistes vastus provides insights into silica biomineralization. R. Soc. Open Sci. 2023 Jun; 10(6): 230423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSará M, Sará A, Nickel M, et al.: Three new species of Tethya (Porifera: Demospongiae) from German Aquaria. Stuttg Beitr Naturkd. 2001 Jan 1; 631: 1–16.\n\nMills DB, Francis WR, Vargas S, et al.: The last common ancestor of animals lacked the HIF pathway and respired in low-oxygen environments. elife. 2018 Feb 6; 7: e31176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlensburg SB, Garm A, Funch P: The contraction-expansion behaviour in the demosponge Tethya wilhelma is light-controlled and follows a diurnal rhythm. J. Exp. Biol. 2022 Dec 7; 225(24): jeb244751. PubMed Abstract \| Publisher Full Text\n\nFrancis W, Eitel M, Vargas S, et al.: The genome of the contractile demosponge Tethya wilhelma and the evolution of metazoan neural signalling pathways. bioRxiv. 2017; p. 120998. Publisher Full Text\n\nEllwanger K, Nickel M: Neuroactive substances specifically modulate rhythmic body contractions in the nerveless metazoon Tethya wilhelma (Demospongiae, Porifera). Front. Zool. 2006 Apr 27; 3: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRivera AS, Hammel JU, Haen KM, et al.: RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria. BMC Biotechnol. 2011 Jan 1; 11(1): 67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHammel JU, Herzen J, Beckmann F, et al.: Sponge budding is a spatiotemporal morphological patterning process: Insights from synchrotron radiation-based x-ray microtomography into the asexual reproduction of Tethya wilhelma. Front. Zool. 2009 Jan 1; 6(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEllwanger K, Eich A, Nickel M: GABA and glutamate specifically induce contractions in the sponge Tethya wilhelma. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 2007 Jan 1; 193(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nFortunato A, Taylor J, Scirone J, et al.: Sponges are highly resistant to radiation exposure and cancer. bioRxiv. 2021 [cited 2024 Mar 20]; p. 2021.03.17.435910. Publisher Full Text\n\nVargas S, Caglar C, Büttner G, et al.: Slime away: a simple CTAB-based high molecular weight DNA and RNA extraction protocol for “difficult” invertebrates.2021 Jul 26 [cited 2022 Dec 23]; Reference Source\n\nBelton JM, McCord RP, Gibcus JH, et al.: Hi-C: a comprehensive technique to capture the conformation of genomes. Methods. 2012 Nov; 58(3): 268–276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPutnam NH, O’Connell BL, Stites JC, et al.: Chromosome-scale shotgun assembly using an in vitro method for long-range linkage. Genome Res. 2016 Mar; 26(3): 342–350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoetzer M, Pirovano W: SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information. BMC Bioinform. 2014 Jun 20; 15: 211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo R, Liu B, Xie Y, et al.: SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012 Dec 27; 1(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeisenfeld NI, Kumar V, Shah P, et al.: Direct determination of diploid genome sequences. Genome Res. 2017 May; 27(5): 757–767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu BH, Xiao J, Xue W, et al.: P_RNA_scaffolder: a fast and accurate genome scaffolder using paired-end RNA-sequencing reads. BMC Genomics. 2018 Mar 2; 19(1): 175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatthey-Doret C, Baudry L, Cournac A, et al.: koszullab/hicstuff: Use miniconda layer for docker and improved P(s) normalisation.2020. Reference Source\n\nJain M, Olsen HE, Paten B, et al.: The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 2016 Nov 25; 17(1): 239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuan J, Li H: Fast and accurate long-read assembly with wtdbg2. Nat. Methods. 2019 Dec 9; 17: 155–158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018 Sep 15; 34(18): 3094–3100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang DD, Li F, Kirton E, et al.: MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ. 2019 Jul 26; 7: e7359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWörheide G, Francis W: PalMuc/2Tethya_genomes. Dataset for: The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae).2024 [cited 2024 May 15]. Reference Source\n\nRio DC, Ares M Jr, Hannon GJ, et al.: Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb. Protoc. 2010 Jun; 2010(6): pdb.prot5439. Publisher Full Text\n\nHaas BJ, Papanicolaou A, Yassour M, et al.: De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 2013 Aug; 8(8): 1494–1512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrůna T, Hoff KJ, Lomsadze A, et al.: BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database. NAR Genom. Bioinform. 2021 Mar; 3(1): lqaa108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoff KJ, Stanke M: WebAUGUSTUS—a web service for training AUGUSTUS and predicting genes in eukaryotes. Nucleic Acids Res. 2013 May 21; 41(W1): W123–W128. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "295816", "date": "12 Jul 2024", "name": "Bernie Degnan", "expertise": [ "Reviewer Expertise Marine biology", "sponges", "genomes", "transcriptomes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWoerheide et al. provide a strong rationale for the sequencing, assembly and analysis of the genomes of two congeneric demosponges, Tethya wilhelma and T. minuta, both which are readily found in public marine aquaria. Using a combination long and short read sequencing and Dovetail HiC/Chicago sequencing, the authors substantially improved the original T. wilhelma genome assembly. The T. minuta assembly, based on long and short reads only, is of sufficient quality for comparison with T. wilhelma genome.\nSuggested revisions: 1.The authors should include details about how the sponge DNA was procured by Dovetail for HiC/Chicago as this could be useful for the sequencing of other sponge and marine invertebrate genomes. 2. Several of the accession numbers in Table 1 can not be traced in NCBI - please update. 3. The synteny plot comparing the two Tethya genomes (Fig. 2) is difficult to interpret and should be improved/reordered to allow better direct comparison of congener scaffolds.\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? Partly\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Partly", "responses": [ { "c_id": "12088", "date": "01 Aug 2024", "name": "Gert Wörheide", "role": "Author Response", "response": "Dear Professor Degnan, thank you very much for your constructive comments on our manuscript to which we respond point by point below: 1. The authors should include details about how the sponge DNA was procured by Dovetail for HiC/Chicago as this could be useful for the sequencing of other sponge and marine invertebrate genomes. Authors reply: Unfortunately, we do not have information how DNA was procured by Dovetail. As stated, we sent them four whole sponges and they extracted chromatin for Chicago and Hi-C according to their proprietary protocols. We then only received the data, without any information on wet-bench protocols. We contacted Dovetail in this matter but have yet to receive a reply. 2. Several of the accession numbers in Table 1 can not be traced in NCBI - please update. Authors reply: The accession number can all be traced in the European Nucleotide Archive (ENA), to which the data was submitted (we double checked all entries). We have, unfortunately, no influence on the timing of data exchange between partners in the International Nucleotide Sequence Database Collaboration (INSDC), where both ENA and NCBI participate. We have now included the direct link to the ENA browser to ease access. https://www.ebi.ac.uk/ena/browser/view/ 3. The synteny plot comparing the two Tethya genomes (Fig. 2) is difficult to interpret and should be improved/reordered to allow better direct comparison of congener scaffolds. Authors reply: Thank you very much for this great suggestion, we have now reordered the plot and highlight some rearrangements in boxes in the plot. We also now provide a much more detailed explanation in the figure caption, i.e., Figure 2: Synteny plot of Tethya wilhelma versus Tethya minuta. Each point represents a homologous gene between the two species. Bars parallel to each axis show the scaffold size. Scaffolds are arranged from longest to shortest in T. wilhelma, with the scaffolds in T. minuta sorted to match the T. wilhelma scaffold with the most homologs. Several rearrangements are evident, some are shown in the orange, blue, and red boxes, which could result from translocations, inversions on currently incomplete scaffolds, or misassemblies." } ] }, { "id": "295815", "date": "13 Jul 2024", "name": "Eric Bautista-Guerrero", "expertise": [ "Reviewer Expertise I'm an expert in integrative taxonomy and molecular phylogeny of marine sponges. I believe that the genome of these two sponges will contribute significantly to our understanding of how this group of invertebrates responds to climate change." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments to the Author\nThis note represents a significant contribution to the field of sponge biology, advancing our understanding of this organism and providing valuable metagenomic resources. It proposes the use of two species of sponge that can be easily maintained long-term in ex situ culture. The sponges in question are proposed as model systems for studying the responses of this invertebrate to the effects of climate change. Furthermore, the sample set is remarkable, and the sequencing and bioinformatics techniques used to improve the draft genome are noteworthy.\nOverall, the Genome note presents a high-quality bioinformatic analysis, and it is recommended that it be accepted pending consideration of minor revisions as detailed below.\nKeywords\n1. First line– Please use italics for the following: Tethya wilhelma, Tethya minuta.\nMethods\n2. DNA extraction: it would be helpful if a brief explanation the modifications to the CTAB method.\n3. In the sentence: “Fragment size was controlled on a 1.5 agarose gel”…., add the percentage should be added after 1.5 (1.5%).\n4. Figure 2: Axes of the graph, scientific name of the sponges in italics.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [ { "c_id": "12089", "date": "01 Aug 2024", "name": "Gert Wörheide", "role": "Author Response", "response": "Dear Professor Bautista-Guerrero, Thank you very much for your constructive comments on our manuscript to which we respond point by point below: 1. First line (Keywords) – Please use italics for the following: Tethya wilhelma, Tethya minuta. Authors reply: Thanks, correct. However, in the Word File I downloaded for the revision the species names in the keywords are correctly formatted in italics, so this must be a typesetting issue on publisher’s side. I will alert them to the fact. 2. DNA extraction: it would be helpful if a brief explanation the modifications to the CTAB method. Author’s reply: Thank for this comment, although the modifications are detailed in the protocol (citation 26) at protocols.io we have now included the following sentence: The modification concerned the addition of Potassium acetate (KOAc, Sigma-Aldrich, Germany, Cat. Nr. 791733) in step no. 5 of the protocol 26. 3. In the sentence: “Fragment size was controlled on a 1.5 agarose gel”…., add the percentage should be added after 1.5 (1.5%). Author’s reply: Thanks for picking this up, we have added the % after 1.5 4. Figure 2: Axes of the graph, scientific name of the sponges in italics. Author’s reply: Thanks, and sorry for this oversight, this has now been corrected in the revised Figure 2 (see reply to comment 3 by Bernie Degnan (Reviewer 1)." } ] } ]	1	https://f1000research.com/articles/13-679
https://f1000research.com/articles/12-774/v1	03 Jul 23	{ "type": "Research Article", "title": "Demographic status and training needs of aesthetic plastic surgeons in government and private hospitals: evidence from Guangdong, China", "authors": [ "Yanhua Yi", "Zhifeng Wang", "Guijie Hu", "Shiwei Zhao", "Yongbin Li", "Bo Chen", "Zhen Xiang", "Qiaojun Zhang", "Wei Lu", "Jian Liu", "Yongping Xue", "Hongmian Li", "Cimin Wu", "Wuxiang Shi", "Zhenyu Gong", "Yanhua Yi", "Zhifeng Wang", "Guijie Hu", "Shiwei Zhao", "Yongbin Li", "Bo Chen", "Zhen Xiang", "Qiaojun Zhang", "Wei Lu", "Jian Liu", "Yongping Xue" ], "abstract": "Purpose: This study aims to survey the demographic status of aesthetic plastic surgeons in an economically developed region and to investigate their continuing training needs in contents, training methods, and barriers in private and government hospitals.\n\nMethods: A cross-sectional survey with a self-administered questionnaire was conducted from January to December, 2022 in Guangdong, China. In-depth interviews were conducted with key informants to gain insights on the current demographic status. Demographic data and training needs assessment were collected and compared with the chi-square test, Fisher’s test, Mann-Whitney U-test using R software. Results: The disparity of surgeons’ demographic data between private and government hospitals was small. Over 60% of practicing aesthetic plastic surgeons transferred from other specialties, and one third of them had less than three years of working experience. Half of surgeons attended training less than three times with an affordable expense of 1000-5000RMB. Almost 80% of them had strong willingness to attend a continuing training program. They prefer to attend further study in a tertiary hospital, with a short topic-focused training course and operation demonstrations. Their favorable training contents were rhinoplasty, eye surgery, and new technologies. Our questionnaire survey revealed the likelihood that the surgeons had a graduate degree in government hospitals outweighed those in private hospitals (P<0.05). Results also showed that the government hospitals focused more on repair and reconstructive surgery and academic research, whereas, private hospitals focused on market needs and were more profit-driven, where the organizational needs had influence on their different training needs for aesthetic plastic surgeons. Conclusion: It would be favorable to take the demographic status of aesthetic plastic surgeons and organizational needs into consideration in designing a continuing training program in plastic surgery.", "keywords": [ "Aesthetic plastic surgeon", "demographic status", "continuing medical training", "needs assessment", "China" ], "content": "Introduction\n\nRapid economic development and “selfie culture” have driven the Chinese plastic surgery market to grow extremely fast in recent years.1,2 It is expected that the total amount of spending on plastic procedures has increased up to approximately 122 billion US dollars by 2019, which ranks third in the world following United States and Brazil.3 As a result, the past decade has witnessed drastic development of private aesthetic plastic hospitals, owing to high demand for all kinds of cosmetic plastic surgeries.4 Meanwhile, surgical doctors area transferring their original practicing specialty into aesthetic plastic surgery after obtaining a training certificate from a tertiary government hospital recognized by the provincial health commission. The government hospitals serve as a training base for medical students who pursue a master degree or PhD degree in plastic surgery and also play pivot roles in continuing training for these doctors who’ve switched from other surgical specialties.5 Private hospitals tend to develop even faster than government hospitals due to more flexibility in management and favorable government policies. The total number of aesthetic plastic procedures in private hospitals has exceeded that of government hospitals.6 Our first hypothesis is that the difference of demographic status between aesthetic plastic surgeons in government hospitals and private hospitals would have different continuing medical training needs.\n\nIt has been documented that continuing medical training programs are significant to improve their serving quality for health professionals.7,8 There have been various training programs available for the aesthetic plastic surgeons in recent years, which include conference sessions, further clinical education, operation demonstrations, and online training courses. But almost all those programs are organized and dictated by hospitals or organizations, the needs of learners have rarely been assessed.9 As Goldstein training mode suggests that training candidates, training contents, organizational requirements be the three important factors while in designing a good health training program.10 Our second hypothesis is the difference between private and government hospital in medical service would lead to different training needs.\n\nObjectives: To date, there is a paucity of data investigating the current demographic status of aesthetic plastic surgeons both in government hospitals and private hospitals in China, and their respective needs in continuing training. To test the above two hypotheses, this study aims to survey the demographic status of aesthetic plastic surgeons from both governmental and private hospitals, and investigate their continuing medical training needs in contents, methods, and barriers within.\n\n\nMethods\n\nEthical approval for this study was obtained from the Ethics Committee of the Guilin Medical University Hospital (approval number: 21-216). Since this research is carried out in the field of continuing medicaleducation (CME) program or in a workshop, informed verbal consent was obtained from all participants.\n\nA mixed study combining a self-administered questionnaire survey with CME participants and in-depth interview with key informants was used.\n\nThe study site was conducted from January to December, 2022 in Guangdong, China. As a coastal province in South China, Guangdong’s GDP in 2021 surpassed 1.5 trillion US dollars, which ranked first in China.11 In this developed economic region, it is estimated that there are more than ten government hospitals with aesthetic plastic departments and two hundred private plastic hospitals with almost eight hundred aesthetic plastic surgeons in total.\n\nVarious CME key informants were identified in consultation with specialists at the department of plastic and aesthetic surgery in Nanfang hospital, Southern Medical University, which is the organizer for many CME programs. This list was expanded after the initial interviews, as the earlier interviewees suggested more informants. Recruitment stopped after the data were considered to be saturated by the researchers. The final informants were the directors of the plastic departments, the heads of medical associations, CME providers (affiliated hospitals of universities), CME users (plastic surgeons). The survey population of CME users included doctors, graduate students when they were attending a continuing medical education program or in a workshop. However, participants who had been employed for less than six months or would retire within a year were excluded.\n\nBased on literature reviews and experts’ consultation, we adapted a questionnaire which was reviewed by a statistician, two epidemiologists, four dermatologists, and two plastic and aesthetic surgeons to ensure its validity. The questionnaire included their demographic information on plastic surgeons, their frequency in attend CME, their preferred contents, type of CME, an barriers and facilitators in attending CME. A small pilot study was conducted to verify suitability of the questions before survey.\n\nIn-depth interviews were conducted with multiple key informants, including the directors of the plastic departments, the heads of medical associations, CME providers (affiliated hospitals of universities), CME users (plastic surgeons). The interview topics included comments on the current status of aesthetic plastic surgeons, current continuing training programs, potential problems, and solutions for the improvement. All interviews were taped and transcribed verbatim for thematic analysis.\n\nAll data analyses were performed using R version 3.5.2 and EpiDisplay package. Categorical data were given as counts and percentages, to compare the difference in demographic information and training needs assessment, the chi-square test, Fisher’s test, were used as appropriate for categorical variables. Mann-Whitney U-test was use to compare the knowledge gap for major aesthetic surgical procedures between surgeons in private and government hospitals. Statistical significance was set at 5%.\n\n\nResults\n\nDemographic data of aesthetic plastic surgeon 290 out of 356 attendees completed the questionnaire survey, the responding rate was 81.5%. 240 surgeons were practicing aesthetic plastic surgery after excluding those cosmetic dermatologists, and cosmetic doctors in traditional Chinese medicine.\n\nShown in Table 1, the distribution of gender, age groups, and years of practice, professional technical titles, and original working department among the 144 surgeons from government hospitals and 96 from private hospital was nearly the same. In general, over sixty percent of the surgeons were under 40 years of age, one third of the surgeons had less than three years of working experience with junior professional technical titles. Almost two thirds of surgeons’ original working departments were not aesthetic plastic surgery. The distribution of educational degree between these hospitals was statistically different. There were more doctors with a graduate degree in government hospitals.\n\nTable 2 summarized the current training status and study objectives for surgeons. Overall, there were some similarities between surgeons of government and private hospitals, half of the surgeons attended training less than three times with an affordable expense of 1000-5000RMB, surgeons in private hospitals were more likely to spend more than 10,000 RMB in training. The majority of them had strong willingness to attend training to improve their practical skills, gain clinical experience, and learn latest technologies.\n\nTable 3 concluded the surgeons’ favorable training methods and reported obstacles. Further study in a tertiary hospital, short training course focusing in specific topic by corresponding experts, and operation demonstration were regarded as the top three favorable choices. The number one obstacle was “nobody replaces me at work”, however, training expenses was the second obstacle for doctors in governmental hospitals and the third one for doctors in private hospital, in converse, “I cannot learn the skills I want” was second obstacle and third one for doctors in private and government hospital respectively.\n\nTable 4 summarized their interest in major surgical procedures and self-assessed knowledge gap. For surgeons in private hospitals, 69.2% of them intended to learn rhinoplasty, and eye plastic surgery. Fat transplant was their third interesting procedures, which coincided with their self-assessed knowledge gap. For surgeons working in government hospitals, their top three interesting surgical procedures were eye plastic surgery, rhinoplasty, and minimally invasive plastic surgery, in terms of knowledge gap, they believed that they had more knowledge gap in external genitalia, perineal surgery, rather than eye plastic surgery.\n\n* Knowledge gap, was calculated by the expected value minus the current assessed value, a five-point Likert scale.\n\nContinuing medical education programs are under the management and organization of medical associations at municipal or provincial levels. Doctors in government hospitals are required to abstain a minimum credit by attending different continuing education programs. However, the doctors in private hospitals are not compulsory required to get the credits. In-depth interviews revealed that most continuing training conferences were co-organized by government and private hospitals. As non-profit medical organizations, the department of aesthetic plastic surgery in most government hospitals, were merged with burn surgery, therefore, they focused more on their social responsibility in repair and reconstructive surgery. Meanwhile, as a base to train medical students, government hospitals paid more attention on academic development and research on basic and clinical technologies. By contrast, private aesthetic plastic hospitals were for-profit originations and laid more emphasis on improving customers’ experience. Since aesthetic plastic procedure were not covered by insurance schemes, customers were more likely to visit private hospitals for operations owing to more comfortable environment and individualized services. These organizational differences between government hospitals and private hospitals determined the different requirements of aesthetic plastic surgeons in their respective institutions, reflecting their needs difference in continuing medical training.\n\n\nDiscussion\n\nThis study suggested that after decades of rapid development of Chinese plastic surgery, the demographic data in private hospitals were quite similar to that of the government hospitals in such an economic developed province. In general, the plastic surgeons were young, with comparatively limited experience. In addition, almost two-thirds of them transferred from other specialties. The only difference was almost half of the doctors in government hospitals had a graduate degree, whereas, a quarter of them had a graduate degree in private hospitals. From the demographic data we concluded that it would be imperative to reinforce continuing training for aesthetic plastic surgery, meanwhile, considering the difference in demographic status.\n\nIn this study, the fact that half of the surgeons attended continuing training no more than three times in a year with limited affordable expense had great significance on planners in designing programs. Although most surgeons preferred to further their studies in a tertiary hospital, the chances were very limited.12,13 This study suggested that a short course focused on specific topic with operation procedures demonstrations by experts would be a favorable training method, a similar finding to other research in surgical education.14 Meanwhile, more attention should be laid on skills improvement in designing training curriculum, and with more consideration of reasonable price and flexible time for attendees.\n\nThis study also revealed a fact that rhinoplasty and new technologies such as fat transplant, minimally invasive plastic surgery were greatly concerning procedures for surgeons either in government hospitals or in private hospitals. To a certain extent, these training needs reflect the needs of the aesthetic market. Since the study site is located in southern China, and there is a big percentage of population with low nasal deformity, the focus on rhinoplasty has also increased accordingly. Minimally invasive cosmetic surgery has grown significantly in the world in recent years, and fat grafting technology is a hot spot in the current market in facial rejuvenation and breast augmentation. The results of self-assessed knowledge gap showed surgeons in government hospitals intend to acquire more continuing medical training in genitoperineal surgery, a newly popular surgical procedure in recent years while the surgeons in private hospitals believed more improvement should be made on plastic surgery for eyes, since blepharoplasty is the most popular procedure in China. The difference in training objectives was due to the different customer groups and social roles for government and private hospitals. Specifically, the government hospitals also took social responsibility for repairtive and reconstructive surgery, while, the private hospitals were more market-oriented, mainly focusing on aesthetic plastic surgery.\n\nThis study tested the hypothesis that the demographic difference for aesthetic plastic surgeons between government and private hospitals were getting narrower. However, the organizational difference had influence on their respective training needs, which coincided with the Goldstein training mode.12,13\n\n\nConclusion\n\nThe demographic disparity of aesthetic plastic surgeons between government hospitals and private hospitals was small. Continuing medical training is very important to improve the training and education of the large number of young aesthetic plastic surgeons in China. To design the training curriculum focused on practical skills, especially a short training course elicited by experts in rhinoplasty, eye surgery, and new technologies such as fat transplant, minimal invasive plastic surgery would be favorable. Meanwhile, the demographic status of aesthetic plastic surgeons and organizational needs should be taken into consideration.", "appendix": "Data availability\n\nDANS-EASY: DEMOGRAPHIC STATUS AND TRAINING NEEDS OF AESTHETIC PLASTIC SURGEONS IN GOVERNMENT AND PRIVATE HOSPITALS: EVIDENCE FROM GUANGDONG, CHINA, https://doi.org/10.17026/dans-2cu-x92u. 14\n\nThis project contains the following underlying data:\n\n• Demographic status and training needs of aesthetic plastic surgeons in government and private hospitals-evidence from Guangdong China.csv\n\nDANS-EASY: DEMOGRAPHIC STATUS AND TRAINING NEEDS OF AESTHETIC PLASTIC SURGEONS IN GOVERNMENT AND PRIVATE HOSPITALS: EVIDENCE FROM GUANGDONG, CHINA, https://doi.org/10.17026/dans-2cu-x92u. 14\n\nThis project contains the following extended data:\n\n• questionaire and interview frame in Chinese.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nPan S, Yin K, Zhang Y, et al.: The transforming training pathway of plastic and craniofacial surgery in China. J. Craniofac. Surg. 2015; 26: 347–349. PubMed Abstract \| Publisher Full Text\n\nZheng J, Zhang B, Yin Y, et al.: Comparison of Plastic Surgery Residency Training in United States and China. Ann. Plast. Surg. 2015; 75: 672–678. PubMed Abstract \| Publisher Full Text\n\nWei L: Selling Cosmetic Surgery and Beauty Ideals: The Female Body in the Web Sites of Chinese Hospitals. Women’s Stud. Commun. 2012; 35: 68–95. Publisher Full Text\n\nLi GS, Dong M-M, Liu L-B, et al.: Ethical Issues in Chinese Aesthetic Surgery. Aesthet. Plast. Surg. 2014; 38: 994–1000. Publisher Full Text\n\nZheng J, Zhang B, Yin Y, et al.: Comparison of Plastic Surgery Residency Training in United States and China. Ann. Plast. Surg. 2015; 75: 672–678. PubMed Abstract \| Publisher Full Text\n\nJin R, Luo X, Wang X, et al.: Complications and Treatment Strategy After Breast Augmentation by Polyacrylamide Hydrogel Injection: Summary of 10-Year Clinical Experience. Aesthet. Plast. Surg. 2018; 42: 402–409. PubMed Abstract \| Publisher Full Text\n\nSong K, Scott A, Sivey P, et al.: Improving Chinese primary care providers’ recruitment and retention: a discrete choice experiment. Health Policy Plan. 2015; 30: 68–77. PubMed Abstract \| Publisher Full Text\n\nSchneider LF, Barr J, Saadeh PB: A nationwide curriculum analysis of integrated plastic surgery training: is training standardized? Plast. Reconstr. Surg. 2013; 132: 1054e–1062e. Publisher Full Text\n\nYi Y, Chongsuvivatwong V, Sriplung H, et al.: Unmet need in continuing medical education programs for rural Chinese township health professionals. J. Educ. Eval. Health Prof. 2015; 12: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavid JA, Rifkin WJ, Saadeh PB, et al.: Assessing the Value of a Multimedia-Based Aesthetic Curriculum in Plastic Surgery Residency: A Single-Center Pilot Study. Aesthet. Surg. J. 2018; 38: NP216–NP224. Publisher Full Text\n\nXiaowen F: An Evolution Analysis of Regional Economic Development Disparities in Guangdong Based on the FPCA. Studies on China’s Special Economic Zones 3. Springer; 2020; 103–113.\n\nAriasSantiago S, GutiérrezSalmerón MT, BuendiaEisman A, et al.: A comparative study of dyslipidaemia in men and women with androgenic alopecia. Acta Derm. Venereol. 2010; 90: 485–487. PubMed Abstract \| Publisher Full Text\n\nGoldstein RF, Boyle JA, Lo C, et al.: Facilitators and barriers to behaviour change within a lifestyle program for women with obesity to prevent excess gestational weight gain: a mixed methods evaluation. BMC Pregnancy Childbirth. 2021; 21: 569. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDr Yi YH : Demographic status and training needs of aesthetic plastic surgeons in government and private hospitals: evidence from Guangdong, China. DANS; 2023. Guilin Medical University. Publisher Full Text" }	[ { "id": "195192", "date": "14 Aug 2023", "name": "Leonard Knoedler", "expertise": [ "Reviewer Expertise Plastic 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\nI commend the authors on an interesting and well-written manuscript investigating the demographic status and training needs of aesthetic plastic surgeons in government and private hospitals. In further detail, the authors elucidated the needs and demographics of aesthetic plastic surgeons in Guangdong, China.\nPlease make the following changes to the manuscript:\n1. Please rework the wording and grammar.\n\n2. Please include a LIMITATIONS section in your discussion. While your work might be of interest to the readership of F1000Research, this work is limited to Guangdong. Therefore, further research is warranted to assess the transferability of your findings in a nationawide/worldwide context.\n3. Please add the following references to provide a broader picture of current trends in plastic surgery. This field is branching out rapidly, exploring new fields and addressing novel challenges. I find it, therefore, crucial to state how broad and diverse the field of plastic surgery is.\na) DOI: 10.1007/s00266-022-02990-9 b) DOI: 10.1016/j.bjps.2022.08.059 c) DOI: 10.1097/SCS.0000000000009106\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10129", "date": "30 Aug 2023", "name": "Yanhua Yi", "role": "Author Response", "response": "I am the first author of this manuscript. I appreciate very much for the reviewer's comments. Although Guangdong province is one of the most economicly developed province in China, and largest in scale in plastic and easthetic surgery, the human resources of plastic surgery in Guangdong is a good representative of China. However, the limitations of this research in evident, we should be careful in generalization. Plastic surgery is changing over time. I appreciate the reviewer's several interesting references suggested." } ] }, { "id": "229131", "date": "16 Jul 2024", "name": "Denis Valente", "expertise": [ "Reviewer Expertise Plastic surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is suitable for indexing. I would like to highlight some points:\nPurpose: The study's aim is clearly stated, providing context for the research. Methods: The methodology is well-described, including the use of a cross-sectional survey, in-depth interviews, and statistical tests. Results: The results are detailed and informative, providing insights into the demographic data of surgeons and their training needs. Discussion: The discussion effectively summarizes the findings and suggests practical applications for the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-774
https://f1000research.com/articles/13-204/v1	21 Mar 24	{ "type": "Study Protocol", "title": "How effective is dentin autograft for socket preservation and implant site preparation: A systematic review protocol.", "authors": [ "Karthik Sivaraman", "Eti Rajwar", "Aditi Chopra", "George Cherukara", "Shubhankar Mehrotra", "Namrata Datta", "Bindhu Koshy", "Karthik Sivaraman", "Eti Rajwar", "George Cherukara", "Shubhankar Mehrotra", "Namrata Datta", "Bindhu Koshy" ], "abstract": "Background Socket preservation is a surgical procedure aimed at preserving the dimensions of the alveolar bone following tooth extraction. It is performed by filling the extraction socket with bone graft material with or without a barrier membrane. Recently, dentine obtained from extracted teeth has been tried as an autograft for socket preservation. Studies have compared the use of dentin to other bone grafts, however, systematic reviews evaluating the efficacy of dentin for socket preservation are limited. Hence, this systematic review protocol is proposed to generate evidence on the efficacy of dentin as a viable alternative to other bone graft materials for socket preservation.\n\nMethods This systematic review protocol was prepared according to the Methodological Expectations of the Cochrane Intervention Reviews (MECIR) guidelines. It will be conducted using the Cochrane Handbook for Systematic Review of Interventions. PubMed, Scopus, Web of Science, EMBASE, Epistemonikos, Cochrane Central, and EBSCO databases and clinical trial registries, will be searched for all randomized controlled trials (RCTs) and non-randomized studies that have used autologous dentin graft (either in particulate/putty, or/matrix form) for socket preservation. The radiographic and clinical assessment of bone and soft tissue healing of the preserved sockets along with patient-related outcomes following surgery will be assessed. The risk of bias assessment of the RCTs and Non-RCTs will be assessed using the ‘Cochrane Risk of Bias assessment tool (ROB II) and ROBINS-I respectively. The certainty of evidence will be assessed by the GRADE approach.\n\nDiscussion This evidence is important for dental clinicians and the public to make an informed decision when choosing graft material for socket preservation. The extracted teeth are considered biological waste; however, this evidence provides scope for using a less invasive autograft for bone regenerative procedures.\n\nSystematic review registration PROSPERO: CRD42021201958 (Registered on 15/02/2021).", "keywords": [ "Dentin", "Bone", "Bone graft", "Implants", "Tooth Extraction", "Socket preservation", "Regeneration", "Healing", "Autograft", "Periodontal surgery" ], "content": "Introduction\n\nDental implants are increasingly being used as a promising modality for replacing missing teeth with superior biological, functional, and aesthetic properties.1 However, one of the important requirements for placing implants is the presence of favorable volume and architecture of the available bone at the time of placement. Many times, adequate bone height and width may not be present to place an implant owing to resorption of the alveolar ridge following extraction.2–5 Approximately 29 to 63% of horizontal bone loss and 11 to 22% of vertical bone loss occur within the first six months following tooth extraction.6 The crest of the alveolar ridge also shifts lingually with more bone loss occurring in the buccolingual dimension (4.5 to 6.1 mm) compared to mesiodistal dimensions.7–14 This reduction in bone volume and thinning of the buccal cortical plates affects the overlying soft tissues, and in turn affects the esthetic, phonetic, and functional outcomes after prosthetic rehabilitation.10–14 Studies have noted that a loss of 2.46 to 4.56 mm ridge width occurs at sites without socket preservation compared to a reduction of 1.14 to 2.5 mm in preserved socket sites.15–25 Additionally, preserving sockets immediately following extraction reduces the need for additional ridge augmentation procedures at the time of implant placement by five times when compared to unassisted socket healing.26–28 Studies have also noted that approximately 20.8 % of non-preserved sockets need additional grafting for implant placement compared to only 9.9 % of preserved sockets.28–35 Thus, to prevent the inevitable resorption of the alveolar ridge, reduce the physiologic bone loss, and retain the volume and quality of bone and associated soft tissue profile, sockets are preserved with bone grafts with and without barrier membranes.15–24\n\nSockets can be preserved with either an autograft, xenograft, allograft, or alloplasts. Among all these bone grafts, autografts have the highest bone regenerative potential.20–22 Autografts can be harvested from edentulous ridges, extraction sockets, mandibular ramus, symphysis, maxillary tuberosity, tibia, iliac crest, and calvarium.20 However, procuring autograft is difficult and requires superior clinical skills. Additionally, many patient-related complications, such as post-operative infection, root resorption, ankylosis, and increased patient morbidity have been noted with the use of autografts.20–24 To overcome these limitations novel bone graft materials with osteogenic potential are being researched.\n\nRecently, whole tooth, enamel, and dentin have been tried as autograft material for various surgical procedures.23–42 Among these dentin autografts are being used owing to their high osteoinductive potential. The composition, physical, and chemical properties of dentin are also similar to that of alveolar bone.30 Dentin also contains insulin-like growth factor, bone morphogenetic protein (BMP), and transforming growth factor-beta (TGF-β) which promotes bone regeneration.25–29 It is available in three forms: demineralized dentin matrix, partially demineralized dentin matrix, and un-demineralized dentin. Dentin bone graft is used for various periodontal and oral surgical procedures such as sinus lift, periodontal defects, ridge augmentation, socket preservation for implant placement, and the treatment of peri-implantitis.26–42 However, there are still various uncertainties and limited systematic reviews related to its use for socket preservation compared to other bone graft materials. The PubMed search with the keyword dentin bone graft yielded only six systematic reviews, with none comparing dentin to other bone graft materials for socket preservation. There is limited data that quantitatively substantiates whether sockets preserved with dentin bone graft are better than those preserved with other bone graft material. Additionally, the understanding of the effect of dentin bone graft on the nature of bone healing and soft tissue healing around the preserved sockets, stability of implants placed in the sockets preserved with dentin, and postoperative complications after using dentin are limited. Thus, this systematic review aims to synthesize evidence on the effectiveness and efficacy of dentin autograft as a graft material for socket preservation compared to allograft, alloplast, and xenograft materials. This evidence is crucial for clinicians, patients, and other stakeholder groups in making evidence-based choices regarding the use of dentin as a bone graft material.\n\n\nObjectives\n\nTo assess the clinical and radiographic changes in the height and width (in mm) of the extraction sockets preserved with dentin autograft (particulate/putty) compared to allograft, alloplast, and xenograft materials.\n\n\n\n1. To evaluate and compare the clinical and radiographic change in the quality of bone and soft tissue healing in sockets preserved with dentin bone graft compared to sockets preserved with other bone grafts (allograft, alloplast, or xenograft).\n\n2. To assess and compare the details of the type of dentin bone graft used, method of application, duration of surgical procedure, patients’ compliance, time of surgery with dentin bone graft compared to sockets preserved with other bone graft (allograft, alloplast or xenograft).\n\n3. To assess and report prognosis and any adverse events/effects or postoperative complications associated with the use of dentin autografts for sockets preservation.\n\n\nMethods\n\nThe proposed systematic review will be conducted according to the Cochrane Handbook for the Systematic Review of Interventions,43 Methodological Expectations of the Cochrane Intervention Reviews (MECIR) guidelines44 and the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) checklist.45,48 The systematic review protocol has been registered in the International Prospective Register of Systematic Review PROSPERO with registration number - CRD42021201958 on 15/02/2021. Differences between the protocol and the final review, if any, because of the methodological alterations due to the unavailability of data, will be reported in the final review.\n\nThe eligibility criteria are described using the Participant-Intervention-Comparison-Outcome-Study Design (PICOS) framework, which also indicates the key components of the research question.\n\nTypes of participants\n\nFor the proposed systematic review, studies with healthy participants who are above 18 years of age and have undergone a socket preservation procedure following tooth extraction will be included.\n\nStudies with participants who have or have not received any dental implants, either at the time of socket preservation or following socket preservation will also be included. Participants who have undergone tooth extraction because of any of the following reasons will be included: unrestorable tooth due to caries or fracture; avulsion of a tooth following trauma; root resorption (internal or external); tooth fracture that cannot be restored; endodontic infection or periodontal disease that cannot be addressed with endodontic and periodontal therapy; failure of root canal treatment, a tooth with grade III mobility and more than 50% bone loss, Grade IV furcation defect (based on Glickman classification) with through and through bone defect that cannot be regenerated and needs an extraction.\n\nFollowing exclusion criteria will be followed:\n\n1. Studies with participants who have a medical history such as diabetes, hypertension, cardiovascular, renal diseases, respiratory, neurological disorders, cancer, psychiatric conditions, autoimmune disease, or pregnancy.\n\n2. Studies with participants suffering from conditions or diseases that might impair bone healing or metabolism, for example, osteoporosis, vitamin D deficiency, and use of anti-resorptive therapy such as bisphosphonates.\n\n3. Studies with participants who use tobacco products, areca nut, gutka, or supari, as smoking. The above factors may influence the uptake of the graft material and or dental implants and hence have been used as exclusion criteria.\n\n4. Studies with participants undergoing chemotherapy or radiation therapy as these treatment modalities compromise angiogenesis and impair bone healing.\n\nTypes of interventions\n\nStudies, where the extraction sockets are preserved with any form of dentin bone graft (demineralized, partially demineralized, or not demineralized) in any form (particulate/putty/matrix/gel), size, or application method with or without a barrier membrane will be included. Studies, where the extraction sockets are preserved with dentin (any particle size) and compared to other bone grafts such as other autografts, alloplasts, allografts, and xenografts with a minimum follow-up of three months will be included. Since bone healing requires a minimum of three months, studies providing a three-month follow-up will be included. Studies where implants are placed immediately following socket preservation will also be included. Studies with both infected and non-infected sockets will be included. Studies where the whole tooth was used as a graft material, will not be considered.\n\nTypes of comparators\n\nStudies, where dentin bone graft is compared with any of the following bone graft material, will be included: autografts (enamel; autologous bone graft from any anatomic site such as chin, ramus, maxillary tuberosity; fresh socket, bone chips, osseous coagulum, bone blend); allograft (Demineralized freeze-dried bone graft and freeze-dried bone graft); xenograft (bovine, porcine, coral-based xenograft); and alloplast (hydroxyapatite; plaster of Paris; tricalcium phosphate; calcium phosphosilicate; calcium phosphate; calcium silicate, etc.).\n\nTypes of outcomes\n\nThe following outcomes will be considered in the proposed review: 1) Clinical and radiographic changes in the height and width (in mm) of the socket before and after socket preservation with dentin bone graft compared to other bone graft; 2) Changes in quality of bone following socket preservation; 3) clinical assessment of soft tissue (in terms of gain in the width of keratinized gingiva) following socket preservation; 4) details of the type of dentin bone graft used, method of application, duration of surgical procedure 5) occurrence of any immediate and delayed post-operative complications following socket preservation surgery.\n\nTypes of studies\n\nIn the proposed systematic review, randomized controlled trials (RCTs), the gold standard study design for assessing the effectiveness of an intervention will be used as the preferred study design for inclusion. RCTs with parallel-group, individual participant, or cluster-allocation designs comparing the independent effects of the intervention on the comparison group will all be included. Non-randomized trials such as pre-post (controlled before-after) and quasi-experimental studies will also be included. Case series with more than 10 subjects and a follow of at least three months would be included. Animal studies, case series with fewer than 10 subjects, letters to the editor, and case reports will all be excluded from inclusion. Only manuscripts published in the English language will be included in the proposed systematic review.\n\nContext or setting\n\nStudies conducted in hospitals, dental clinics, university hospitals, and other relevant healthcare facilities, dependent on the country where the study was conducted, will be included in the review. Studies, where treatment was provided by specialists and non-specialists, will be included in the review.\n\nA search strategy was developed using keywords identified from the preliminary search and following agreement between the research team (KS, AC, ER) and a search expert (GC, BK). It included Medical Subject Headings (Mesh) terms, free text terms, and Boolean (AND, OR), as well as the components of the PICO framework. The advanced search strategy for PubMed, one of the databases to be searched is given in Table 1. An electronic search for English language publications in PubMed, Scopus, Web of Science, EMBASE, Epistemonikos, EBSCO (Dentistry and Open Access), and Cochrane CENTRAL databases will be conducted. Additionally, ProQuest, World Clinical Trial Registry, and Clinical Trials Registry of India (CTRI) will be searched for ongoing clinical trials. The corresponding authors for the ongoing clinical trials will be requested to share the results (if available). In addition, the bibliography section of the following journals will be searched for any articles in press: Journal of Periodontology, Journal of Dental Research, Journal of Periodontal Research, Journal of Clinical Periodontology, Journal of Clinical Oral Implant Research, Journal of Implantology, Journal of Clinical Implants and Related Research. We will also check references of the included studies for other relevant articles. Preprint servers such as BioRxiv, Research Square, and MedRxiv will be screened for relevant articles on dentin as bone graft material used in socket preservation. Doctoral theses, conference proceedings, and industry studies published in company publications will also be screened for relevant literature.\n\nThe search results will be managed using the Microsoft Office (MS) Excel software. After the removal of duplicates, four reviewers (KS, AC, SM, ND) will independently select studies using the inclusion and exclusion criteria. The screening process will be performed in two stages. In the first stage, the titles, and abstracts of the studies will be independently screened. Studies that do not qualify according to the eligibility criteria and those with inadequate data or information will be excluded at the title/abstract (Ti/Ab) screening stage. In the second stage, full-text screening of the included studies will be performed independently by the four reviewers (KS, AC, SM, ND). They will also perform hand-searching of the journals listed above and screen the potential articles for inclusion. Any disagreements regarding inclusion will be resolved initially by discussion between the reviewers (GC) and if unresolved the other reviewers (BK, KS) will be consulted for agreement; a majority decision will be adopted. A pre-designed screening protocol will be used to select studies (Table 2).\n\nThree reviewers (KS, AC, ND, SM) will perform data extraction independently, using a pre-designed pilot-tested data extraction sheet. Any disagreements during the extraction process will be resolved initially by discussion between three reviewers, and if still unresolved BK and GC will be consulted. A majority decision will be adopted as final. In case of unpublished and missing information/data, the study authors will be contacted to request the required information. If adequate data are available, the review team will perform additional relevant statistical analysis.\n\nThe outcomes considered in the proposed review are:\n\nPrimary outcomes:\n\ni. Clinical and radiographic assessment of the overall bone volume gain or otherwise, following socket preservation as measured by the gain or otherwise, in the mesiodistal, buccolingual, and apicocoronal dimensions of the socket before and after the intervention (in mm) will be measured. This can be measured by bone-sounding/trans-gingival probing or any radiographic assessment. The changes in the proximal/interdental bone levels at teeth-bounding extraction sites will also be noted.\n\nii. The nature of bone, and quality of bone as assessed by any clinical or radiographic method will be considered. Radiographic assessment of bone volume or quality as measured by changes in the density of the trabecular pattern (no/mm2); changes in the trabecular pattern; ratio of cortical to cancellous bone in terms of Hounsfield unit (HU) in the extraction socket pre- and post-intervention (mm2); or any other techniques reported in respective studies will be considered. The stability of implants in sockets preserved with dentin bone graft compared to other bone grafts is of interest.\n\nIf the clinical method is not used and the radiographic method is used in the studies to provide the change in the dimension of the socket and it does not provide three-dimensional information, the two-dimensional measurement will be recorded. The method of assessment will also be recorded. These outcomes have been chosen as the primary outcomes because the quality and quantity of bone and soft tissue following socket preservation are the most crucial outcomes to define success.\n\nSecondary outcomes:\n\ni. The health of soft tissue around the socket preserved, as measured by the health of the gingival tissues and gain in the amount of keratinized tissue after socket preservation at baseline and post-treatment, as measured by any histomorphometric analysis or manually by any periodontal probe will be noted.\n\nii. Percentage/amount of bone graft remaining in the socket post-intervention (at each recall) reported by surgical re-entry, histology, or radiographic imaging technique will be considered.\n\niii. Presence or absence of any postoperative complications at the time of surgery, immediately after surgery; within 24 hours, or delayed after socket preservation will be considered. Presence or absence of non-healed sockets, delayed socket healing, exposure of barrier membrane, loss of graft material, presence and presence of soft tissue defects on the alveolar ridge; presence or absence of any suppuration, pus discharge, excessive bleeding, dehiscence and opening of the wound, fenestration on the buccal or lingual aspect of the closed wound, recession of soft tissue following surgery, postoperative pain, persistent pain or dysesthesia following treatment as measured by any pain scale such as Visual Analog Scale (VAS), Numeric Rating Scale (NRS), and The Wong-Baker face pain scale following socket preservation are other will be considered.\n\niv. Time required between socket preservation and implant placement; nature of osseointegration if implants were placed at the time of socket preservation will be considered.\n\nv. Health of the tooth used for procurement of dentin; type of dentine used, method of processing of dentin; time of utilization of tooth following extraction; method of storage of dentin, mode of application of dentin (with or without any device) and particle size of dentin graft (if mentioned); use of dentin with or without a barrier membrane will be considered.\n\nThe risk of bias will be assessed using the revised Cochrane Risk of Bias Assessment Tool (ROB2).46,47 Two review authors (KS, AC) will independently assess the risk of bias in the included studies. Any disagreements will be resolved via discussion, if consensus cannot be reached, then a majority decision will be adopted after consulting the other two reviewers (BK, GC). The following domains will be assessed for risk of bias: 1. Randomization process; 2. Bias resulting from deviations from intended interventions; 3. Bias resulting from missing outcome data; 4. Bias resulting from measurement of the outcome; 5. Bias resulting from selection of the reported result; 6. Bias in the identification or recruitment of individual participants within clusters. If cluster-RCTs are included, whether the reported data analysis had appropriately taken account of the aggregate nature of the data will be determined. The identified risks shall be categorized as low risk, high risk, and risk with some concern. The ‘effects of assignment’ will be considered as the effect of principal interest to assess the risk of bias.\n\nQuality assessment of non-randomized studies will be performed using the Risk of Bias In Non-Randomized Studies of Interventions (ROBINS-I).47 As per this tool, bias in a non-randomized trial will be assessed for the following seven domains: 1) Bias due to confounding, 2) Bias in the selection of participants into the study, 3) Bias in the classification of interventions, 4) Bias due to deviations from intended interventions, 5) Bias due to missing data, 6) Bias in measurement of outcomes, 7) Bias in selection of the reported result. The assessed risk will be categorized as low risk, moderate risk, serious risk, and critical risk.\n\nIn the case of continuous data, Mean Difference (MD) and Standard Mean Difference (SMD) with 95% Confidence Interval (CI) will be used to estimate the effects of the intervention. Odds Ratio (OR) and Risk Ratio (RR) with 95% CI will be used for categorical data.\n\nThe participant will be the unit of analysis for individual randomized trials, however, a study cluster will be considered as the unit of analysis for cluster-randomized trials. The impact of data clustering will be considered and the unit of analysis will be adjusted accordingly during analysis.\n\nMeta-analysis will be performed in case homogenous data is obtained from the included studies. I2 statistic will be used to assess statistical heterogeneity, describing the percentage of the variability in effect estimates that is due to heterogeneity rather than chance. Based on the I2 statistic, the level of heterogeneity will be categorized as follows:\n\n• 0% to 40%: might not be important\n\n• 30% to 60%: may represent moderate heterogeneity\n\n• 50% to 90%: may represent substantial heterogeneity\n\n• 75% to 100%: considerable heterogeneity\n\nA fixed-effects model will be used for meta-analysis. A forest plot will be used to report the results of the meta-analysis. A narrative synthesis, with tables and figures for explanation, will be performed when meta-analysis is not possible in case of more than 50% ‘clinical,’ ‘methodological,’ and ‘statistical’ heterogeneity.\n\nIf possible, sub-group analysis will be performed based on the height and width of the socket before and after socket preservation; quality of bone formed before and after socket preservation in dentin compared to other bone grafts; nature of soft tissue before and after socket preservation in dentin compared to other bone graft; post-operative complications following socket preservation.\n\nFunnel plots will be used for the identification of reporting bias. The identification of reporting bias will be done based on the visual assessment of the funnel plot, where a symmetrical funnel plot will be interpreted as the absence of reporting bias and an asymmetrical plot will indicate the presence of reporting bias.\n\nIf sufficient data is available, a sensitivity analysis will be performed to assess the robustness of the results. This will be done using studies with a ‘low risk of bias’.\n\nThe certainty of evidence will be assessed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach. This approach grades evidence for five domains 1) risk of bias, 2) indirectness, 3) inconsistency, 4) imprecision, and 5) publication bias. The level of certainty of evidence will be classified as high, moderate, low, and very low, using the GRADE Pro GDT software, independently by three of the review authors (KS, AC, and ER). Disagreements will be resolved by discussion and in case of non-consensus, the two other authors (BK and GC) will be consulted, and a majority decision taken. The certainty of evidence will be reported for the following seven outcomes 1) Changes in the dimensions (height and width) of the alveolar ridge following grafting; 2) Nature of bone and soft tissue healing following socket preservation; 3) Postoperative complications (presence and absence of pain, dysesthesia, suppuration, marginal bone loss or loss of graft material.\n\nThe review is currently in the full-text screening stage.\n\n\nDiscussion\n\nThe proposed systematic review will assess the quality of the evidence base available regarding the efficacy and efficiency of using dentine bone graft material in socket preservation. The review will help to understand whether dentin has a superior osteogenic potential than other bone grafts and what future complications one should note when using dentin. This will help to understand if dentin is a good alternative to conventional autografts like ramus graft and chin graft which involve many patient-related complications and morbidity.18–20 Since many times tooth is discarded as a biological waste, the utilization of the same tooth for regeneration of bone defect would preclude the need for additional bone graft material. This would help patients to obtain a cheaper, affordable, and relatively easier option compared to other autografts. Hence the evidence is crucial for the patients and clinicians who wish to make informed decisions about choosing dentin autograft, in resource-constrained settings.23–32 We have involved relevant stakeholders such as clinicians, patients, and researchers for the identification of research uncertainties and the formulation of the research question. After consultation with the stakeholders, the need for a non-invasive, cheaper, and more comfortable option for procuring autografts was deemed necessary. We also took feedback from patients in our clinics regarding their experience following surgery where autograft was procured from the chin and ramus. There is a need to explore this research knowledge and explore the efficacy of dentin as bone graft material for socket preservation and at the same it is important to understand the clinical outcomes and patient-related outcomes after surgery using dentin as a bone graft material. The review will also cue the dentist to understand the probable complications associated with dentin bone graft. Thus, the comprehensive evaluation of research and the resulting consolidated up-to-date evidence will help clinicians make evidence-informed decisions while choosing graft material. Additionally, the review will also propose further studies required to address gaps identified in the knowledge created so far on dentine as a graft material. It is hoped that collaborative research with clinicians, scientists, and the industry will be promoted as a result of the planned review. However, one should note that one of the limitations of the proposed review is that the evidence will be based on English language literature only. Although this review aims to explore, the role of dentin as graft material for socket preservation, we would not be able to provide strong evidence on the stability, prognosis, and long-term survival of the implants as these factors have many confounding variables that cannot be addressed and is not within the scope of this review. However, future studies should explore the prognosis and survival rate of implants in sockets preserved in the dentin compared to other bone grafts.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: PRISMA-P checklist for ‘How effective is dentin autograft for socket preservation and implant site preparation: A systematic review protocol.’ https://doi.org/10.6084/m9.figshare.25195910. 48\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe would like to acknowledge Manipal Academy of Higher Education, Manipal, Karnataka, for logistic and technical support.\n\n\nReferences\n\nGarcía-González S, Galve-Huertas A, Aboul-Hosn Centenero S, et al.: Volumetric changes in alveolar ridge preservation with a compromised buccal wall: a systematic review and meta-analysis. Med. Oral Patol. Oral Cir. Bucal. 2020; 25(5): e565–e575. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorjaria KR, Wilson R, Palmer RM: Bone healing after tooth extraction with or without an intervention: a systematic review of randomized controlled trials. Clin. Implant. Dent. Relat. Res. 2014; 16(1): 1–20. Publisher Full Text\n\nBarallat L, Ruíz-Magaz V, Levi PA Jr, et al.: Histomorphometric results in ridge preservation procedures comparing various graft materials in extraction sockets with nongrafted sockets in humans: a systematic review. Implant. Dent. 2014; 23(5): 539–554. PubMed Abstract \| Publisher Full Text\n\nBarone A, Ricci M, Tonelli P, et al.: Tissue changes of extraction sockets in humans: a comparison of spontaneous healing vs. ridge preservation with secondary soft tissue healing. Clin. Oral Implants Res. 2013; 24(11): 1231–1237. PubMed Abstract \| Publisher Full Text\n\nJambhekar S, Kernen F, Bidra AS: Clinical and histologic outcomes of socket grafting after flapless tooth extraction: a systematic review of randomized controlled clinical trials. J. Prosthet. Dent. 2015; 113(5): 371–382. PubMed Abstract \| Publisher Full Text\n\nChan HL, Lin GH, Fu JH, et al.: Alterations in bone quality after socket preservation with grafting materials: a systematic review. Int. J. Oral Maxillofac. Implants. 2013; 28(3): 710–720. PubMed Abstract \| Publisher Full Text\n\nSchropp L, Wenzel A, Kostopoulos L, et al.: Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int. J. Periodontics Restorative Dent. 2003; 23(4): 313–323. PubMed Abstract\n\nChen ST, Darby I: The relationship between facial bone wall defects and dimensional alterations of the ridge following flapless tooth extraction in the anterior maxilla. Clin. Oral Implants Res. 2017; 28(8): 931–937. PubMed Abstract \| Publisher Full Text\n\nVan der Weijden F, Dell’Acqua F, Slot DE: Alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. J. Clin. Periodontol. 2009; 36(12): 1048–1058. PubMed Abstract \| Publisher Full Text\n\nAraújo MG, Lindhe J: Dimensional ridge alterations following tooth extraction. An experimental study in the dog. J. Clin. Periodontol. 2005; 32(2): 212–218. PubMed Abstract \| Publisher Full Text\n\nLópez-Jarana P, Díaz-Castro CM, Falcão A, et al.: Thickness of the buccal bone wall and root angulation in the maxilla and mandible: an approach to cone beam computed tomography. BMC Oral Health. 2018; 18(1): 194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Risi V, Clementini M, Vittorini G, et al.: Alveolar ridge preservation techniques: a systematic review and meta-analysis of histological and histomorphometrical data. Clin. Oral Implants Res. 2015; 26(1): 50–68. PubMed Abstract \| Publisher Full Text\n\nRasperini G, Canullo L, Dellavia C, et al.: Socket grafting in the posterior maxilla reduces the need for sinus augmentation. Int. J. Periodontics Restorative Dent. 2010; 30(3): 265–273. PubMed Abstract\n\nTen Heggeler JM, Slot DE, Van der Weijden GA: Effect of socket preservation therapies following tooth extraction in non-molar regions in humans: a systematic review. Clin. Oral Implants Res. 2011; 22(8): 779–788. PubMed Abstract \| Publisher Full Text\n\nAvila-Ortiz G, Elangovan S, Kramer KW, et al.: Effect of alveolar ridge preservation after tooth extraction: a systematic review and meta-analysis. J. Dent. Res. 2014; 93(10): 950–958. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAvila-Ortiz G, Chambrone L, Vignoletti F: Effect of alveolar ridge preservation interventions following tooth extraction: A systematic review and meta-analysis. J. Clin. Periodontol. 2019; 46(Suppl 21): 195–223. PubMed Abstract \| Publisher Full Text\n\nWillenbacher M, Al-Nawas B, Berres M, et al.: The Effects of Alveolar Ridge Preservation: A Meta-Analysis. Clin. Implant. Dent. Relat. Res. 2016; 18(6): 1248–1268. Publisher Full Text\n\nSittitavornwong S, Gutta R: Bone graft harvesting from regional sites. Oral Maxillofac. Surg. Clin. North Am. 2010; 22(3): 317–330. Publisher Full Text\n\nTroiano G, Zhurakivska K, Lo Muzio L, et al.: Combination of bone graft and resorbable membrane for alveolar ridge preservation: A systematic review, meta-analysis, and trial sequential analysis. J. Periodontol. 2018; 89(1): 46–57. PubMed Abstract \| Publisher Full Text\n\nZouhary KJ: Bone graft harvesting from distant sites: concepts and techniques. Oral Maxillofac. Surg. Clin. North Am. 2010; 22(3): 301–316. PubMed Abstract \| Publisher Full Text\n\nAllegrini S Jr, Koening B Jr, Allegrini MR, et al.: Alveolar ridge sockets preservation with bone grafting--review. Ann. Acad. Med. Stetin. 2008; 54(1): 70–81. PubMed Abstract\n\nNkenke E, Schultze-Mosgau S, Radespiel-Tröger M, et al.: Morbidity of harvesting of chin grafts: a prospective study. Clin. Oral Implants Res. 2001; 12(5): 495–502. PubMed Abstract \| Publisher Full Text\n\nZhang S, Li X, Qi Y, et al.: Comparison of Autogenous Tooth Materials and Other Bone Grafts. Tissue Eng. Regen. Med. 2021; 18(3): 327–341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtieh MA, Alsabeeha NH, Payne AG, et al.: Interventions for replacing missing teeth: alveolar ridge preservation techniques for dental implant site development. Cochrane Database Syst. Rev. 2021; 2021(4): CD010176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUm IW: Demineralized Dentin Matrix (DDM) As a Carrier for Recombinant Human Bone Morphogenetic Proteins (rhBMP-2). Adv. Exp. Med. Biol. 2018; 1077: 487–499. PubMed Abstract \| Publisher Full Text\n\nUm IW, Kim YK, Mitsugi M: Demineralized dentin matrix scaffolds for alveolar bone engineering. J. Indian Prosthodont. Soc. 2017; 17(2): 120–127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoga T, Minamizato T, Kawai Y, et al.: Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size. PLoS One. 2016; 11(1): e0147235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReis-Filho CR, Silva ER, Martins AB, et al.: Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats. Arch. Oral Biol. 2012; 57(5): 469–476. PubMed Abstract \| Publisher Full Text\n\nTabatabaei FS, Tatari S, Samadi R, et al.: Different methods of dentin processing for application in bone tissue engineering: A systematic review. J. Biomed. Mater. Res. A. 2016; 104(10): 2616–2627. PubMed Abstract \| Publisher Full Text\n\nYüceer-Çetiner E, Özkan N, Önger ME: Effect of Autogenous Dentin Graft on New Bone Formation. J. Craniofac. Surg. 2021; 32(4): 1354–1360. PubMed Abstract \| Publisher Full Text\n\nLee JY, Kim YK, Yi YJ, et al.: Clinical evaluation of ridge augmentation using autogenous tooth bone graft material: case series study. J. Korean Assoc. Oral Maxillofac. Surg. 2013; 39(4): 156–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPang KM, Um IW, Kim YK, et al.: Autogenous demineralized dentin matrix from extracted tooth for the augmentation of alveolar bone defect: a prospective randomized clinical trial in comparison with anorganic bovine bone. Clin. Oral Implants Res. 2017; 28(7): 809–815. PubMed Abstract \| Publisher Full Text\n\nRamanauskaite A, Sahin D, Sader R, et al.: Efficacy of autogenous teeth for the reconstruction of alveolar ridge deficiencies: a systematic review. Clin. Oral Investig. 2019; 23(12): 4263–4287. PubMed Abstract \| Publisher Full Text\n\nÖzkahraman N, Balcıoğlu NB, Soluk Tekkesin M, et al.: Evaluation of the Efficacy of Mineralized Dentin Graft in the Treatment of Intraosseous Defects: An Experimental in vivo Study. Medicina (Kaunas). 2022; 58(1): 103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCervera-Maillo JM, Morales-Schwarz D, Morales-Melendez H, et al.: Autologous Tooth Dentin Graft: A Retrospective Study in Humans. Medicina (Kaunas). 2021; 58(1): 56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNadershah M, Zahid TM: Use of Autogenous Dentin Graft in Mandibular Third Molar Extraction Sockets: A Split-Mouth Randomized Double-Blind Study. Int. J. Pharm. Res. Allied Sci. 2019; 8(3): 73–79.\n\nNkenke E, Neukam FW: Autogenous bone harvesting and grafting in advanced jaw resorption: morbidity, resorption and implant survival. Eur. J. Oral Implantol. 2014; 7(Suppl 2): S203–S217.\n\nGharpure AS, Bhatavadekar NB: Clinical Efficacy of Tooth-Bone Graft: A Systematic Review and Risk of Bias Analysis of Randomized Control Trials and Observational Studies. Implant. Dent. 2018; 27(1): 119–134. PubMed Abstract \| Publisher Full Text\n\nMahardawi B, Jiaranuchart S, Tompkins KA, et al.: Efficacy of the autogenous dentin graft for implant placement: a systematic review and meta-analysis of randomized controlled trials. Int. J. Oral Maxillofac. Surg. 2023; 52(5): 604–612. PubMed Abstract \| Publisher Full Text\n\nStumbras A, Kuliesius P, Januzis G, et al.: Alveolar Ridge Preservation after Tooth Extraction Using Different Bone Graft Materials and Autologous Platelet Concentrates: a Systematic Review. J. Oral Maxillofac. Res. 2019; 10(1): e2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVittorini Orgeas G, Clementini M, De Risi V, et al.: Surgical techniques for alveolar socket preservation: a systematic review. Int. J. Oral Maxillofac. Implants. 2013; 28(4): 1049–1061. Publisher Full Text\n\nSánchez-Labrador L, Bazal-Bonelli S, Pérez-González F, et al.: Autogenous particulated dentin for alveolar ridge preservation. A systematic review. Ann. Anat. 2023; 246: 152024. PubMed Abstract \| Publisher Full Text\n\nHiggins JPT, Thomas J, Chandler J, et al.: Cochrane Handbook for systematic reviews of interventions version 6.1 (updated September 2020). London: Cochrane; 2020.\n\nHiggins JPT, Lasserson T, Chandler J, et al.: Methodological Expectations of Cochrane Intervention Reviews. London: Cochrane; February 2021. Version. MECIR Version February 2021 clean final_1.pdf Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSterne JAC, Savović J, Page MJ, et al.: RoB 2: a revised tool for assessing risk of bias in randomised trials. BMJ (Clinical research ed.). 2019; 366: l4898. Publisher Full Text\n\nSterne JA, Hernán MA, Reeves BC, et al.: ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ (Clinical research ed.). 2016; 355: i4919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChopra A: PRISMA-P checklist for dentin bone graft for socket preservation: systematic review. [Dataset]. Figshare. 2024. Publisher Full Text" }	[ { "id": "295738", "date": "08 Jul 2024", "name": "Lydia N. Melek", "expertise": [ "Reviewer Expertise Oral and Maxillofacial surgery with a special interest in dental implantology and grafts. I have several publications on the use of dentin graft." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 protocol is very well-designed with sufficient feedback about the topic and adequately addresses the current knowledge gap in utilizing the dentin graft as an autograft alternative. The study aim is clear and the methods are reproducible. The authors are advised to add a separate subtitle with the research question of the systematic review stated clearly and concisely for the reader. Also, the authors are advised to add the time frame for the studies included in the review. It's mentioned in Table 2 \"year 2000 or after\" but it's better to add it in the manuscript too.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "12091", "date": "01 Aug 2024", "name": "Aditi Chopra", "role": "Author Response", "response": "Firstly I would like to thank the editor and all the reviewers for their valuable suggestions and insightful comments. It has helped us in improving our work. We have tried our best to revise the manuscript as per the comments. The changes are highlighted in yellow. Reviewer 1 Comment 1: The systematic review protocol is very well-designed with sufficient feedback about the topic and adequately addresses the current knowledge gap in utilizing the dentin graft as an autograft alternative. The study aim is clear and the methods are reproducible. The authors are advised to add a separate subtitle with the research question of the systematic review stated clearly and concisely for the reader. Response: We have now added a separate subtitle as the focus question as suggested. Comment 2: Also, the authors are advised to add the time frame for the studies included in the review. It's mentioned in Table 2 \"year 2000 or after\" but it's better to add it in the manuscript too. Response: We have now added the study duration in the text." } ] }, { "id": "295742", "date": "22 Jul 2024", "name": "Marwa Madi", "expertise": [ "Reviewer Expertise Periodontology", "implant 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 study is well written and the methodology is explained in a detailed and organized manner. Since the objective of the review is clinical and radiographic findings as well as soft tissue healing following socket preservation using dentin allograft I think the authors need to exclude all animal studies and include only clinical studies. Strength of the proposal:\nThe proposal addresses a novel approach by investigating the use of dentin autografts for socket preservation, which is an emerging area in dental research. This originality is relevant and significant for students at this level. The proposal is grounded in a well-established scientific premise, highlighting the importance of alveolar ridge preservation and the potential benefits of using dentin as a graft material. The background information provided is comprehensive and supports the rationale for the study. The methodology is robust, following the Cochrane Handbook for Systematic Reviews and adhering to PRISMA-P guidelines. The use of multiple databases for literature search, clear inclusion and exclusion criteria, and detailed data extraction and analysis plan demonstrate feasibility and rigor.\n\nThe weakness of the proposal: Focus on Specific Population:\nThe study focuses on healthy adults, potentially overlooking how dentin autografts might perform in populations with comorbidities or different age groups.\nLimited Scope of Comparative Studies: While the proposal compares dentin autografts with other types of grafts, it does not account for potential variations in outcomes based on patient demographics or specific clinical conditions.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "12092", "date": "01 Aug 2024", "name": "Aditi Chopra", "role": "Author Response", "response": "Reviewer 2 Comment 1: Since the review's objective is clinical and radiographic findings and soft tissue healing following socket preservation using dentin allograft, I think the authors need to exclude all animal studies and include only clinical studies. Response: We agree with the reviewer, We have added these points to the eligibility criteria. Comment 2: The study focuses on healthy adults, potentially overlooking how dentin autografts might perform in populations with comorbidities or different age groups. Response: We thank the reviewer for their valuable insight. We agree that the effect of dentin should be checked in diverse populations to establish its validity and efficacy. Hence, we would like to highlight that we would not exclude studies where dentin was used in populations with co-morbidities. This would even help us to do a subgroup analysis and check. We have added these points to our eligibility criteria. Comment 3: While the proposal compares dentin autografts with other types of grafts, it does not account for potential variations in outcomes based on patient demographics or specific clinical conditions. Response: We agree with the reviewer for this valuable suggestion. Based on the articles we retrieved, we can analyze the clinical outcomes based on variation in variations in demographic and clinical conditions." } ] } ]	1	https://f1000research.com/articles/13-204
https://f1000research.com/articles/12-148/v1	09 Feb 23	{ "type": "Data Note", "title": "The identification of high-performing antibodies for Midkine for use in Western blot and immunoprecipitation", "authors": [ "Riham Ayoubi", "Kathleen Southern", "Carl Laflamme", "NeuroSGC/YCharOS Collaborative Group", "Riham Ayoubi", "Kathleen Southern" ], "abstract": "Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.", "keywords": [ "Uniprot #P21741", "MDK", "Midkine", "antibody validation", "antibody characterization", "Western blot", "immunoprecipitation" ], "content": "Introduction\n\nThe neurotrophic and developmental factor Midkine is a secreted heparin-binding cytokine.1 It mediates its diverse physiological functions by binding to cell-surface proteoglycan receptors via their chondroitin sulfate groups, thereby regulating cell proliferation, migration and differentiation.2–4\n\nMidkine is involved in numerous processes that promote cell growth, and may contribute to the pathogenesis of inflammatory diseases.1,4 Proteomic analyses have found that Midkine is highly enriched in amyloid-beta plaques, indicating its potential as a biomarker and/or therapeutic target for Alzheimer’s Disease (AD).5,6 Mechanistic studies would be greatly facilitated by the availability of high-quality antibodies for Midkine.\n\nHere, we compared the performance of a range of commercially available antibodies for Midkine and identified high-quality antibodies for Western Blot and immunoprecipitation, enabling biochemical and cellular assessment of Midkine properties and function.\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type and knockout cells.7,8 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM”+1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID: SCR_017655). Commercially available HAP1 cells expressed the Midkine transcript at RNA levels above the average range of cancer cells analyzed.9 Parental and MDK knockout HAP1 cells were obtained from Horizon Discovery (Table 1).\n\nMidkine is predicted to be a secreted protein. Accordingly, we collected concentrated culture media from both wild-type and MDK KO cells and used the conditioned media to probe the performance of the antibodies (Table 2) side-by-side by Western blot and immunoprecipitation. The profiles of the tested antibodies are shown in Figures 1 and 2.\n\n* Monoclonal antibody.\n\n Recombinant antibody.\n\nA) Schematic of the protocol used to concentrate the culture media. B) Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot with the indicated Midkine antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies GTX116089 and ab52637, which were titrated to 1/1000 and 1/500, respectively, as the signal was too weak when following the supplier’s recommendations. When suppliers did not recommend dilutions, we tested the antibodies at 1/200. Antibody dilution used: GTX108439 at 1/1000; GTX116089 at 1/100; AF-258-PB at 1/100; MAB2582** at 1/200; MAB2583* at 1/200; NBP2-66948 at 1/500; MA5-32538 at 1/500; ab52637** at 1/500. Predicted band size: 15 kDa. Monoclonal antibody, Recombinant antibody.\n\nConcentrated culture media were prepared as in 1A). Immunoprecipitation was performed using 1.0 μg of the indicated Midkine antibodies pre-coupled to either protein A or protein G magnetic beads. Samples were washed and processed for Western blot with the indicated Midkine antibody. For Western blot, AF-258-PB was used at 1/500, NBP2-66948* at 1/500, MA5-32538 at 1/200 and ab52637 at 1/200. The Ponceau stained transfers of each blot are shown. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate; HC = antibody heavy chain. Monoclonal antibody, *Recombinant antibody.\n\nIn conclusion, we screened Midkine commercial antibodies by Western blot and immunoprecipitation and identified several high-quality antibodies under our standardized experimental conditions.\n\n\nMethods\n\nAll Midkine antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit, anti-mouse and donkey anti-goat antibodies are from Thermo Fisher Scientific (cat. number 65-6120, 62-6520 and A15999, respectively).\n\nCells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201). Cells were starved in DMEM high glucose containing L-glutamate and penicillin/streptomycin.\n\nHAP1 cells (WT and MDK KO) were washed 3× with PBS and starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 × g to eliminate cells and larger contaminants, then for 10 min at 4500 × g to eliminate smaller contaminants.\n\nCulture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 × g for 15 min. The resulting 500 μl of the concentrated media were centrifuged again at 4000 × g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl.\n\nWestern blots were performed as described in our standard operating procedure.10 Western blots were performed with large 10-20% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three more washes with TBST. Membranes were incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).\n\nImmunoprecipitation was performed as described in our standard operating procedure.11 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of Pierce IP Lysis from Thermo Fisher Scientific (cat. number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse and goat antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked overnight at 4°C followed by two washes with the Pierce IP Buffer to remove unbound antibodies. Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail from Thermo Fisher Scientific (cat. number 78446).\n\nStarved HAP1 WT culture media were concentrated as described above. The concentrated culture media were diluted in Pierce IP Lysis Buffer, and 1ml aliquots at 0.3 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP Buffer and processed for SDS-PAGE and immunoblot on 10-20% polyacrylamide gels. Prot-A: HRP was used as a secondary detection system (MilliporeSigma, cat. number P8651) at a dilution of 0.4 μg/ml for an experiment where a rabbit antibody was used for both immunoprecipitation and its corresponding Western Blot.", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Midkine, https://doi.org/10.5281/zenodo.5644321. 12\n\nZenodo: Dataset for the Midkine antibody screening study, https://doi.org/10.5281/zenodo.7530472. 13\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nThe authors would like to thank the NeuroSGC/YCharOS collaborative group for the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, as well as the development of community-agreed protocols. Members of this group can be found below. NeuroSGC/YCharOS collaborative group: Riham Ayoubi, Kathleen Southern, Peter S. McPherson, Aled M. Edwards, Chetan Raina and Carl Laflamme.\n\nAn earlier version of this article can be found on Zenodo (doi:10.5281/zenodo.5644321). 12\n\n\nReferences\n\nMuramatsu T: Structure and function of midkine as the basis of its pharmacological effects. Br. J. Pharmacol. 2014; 171(4): 814–826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurosawa N, Chen GY, Kadomatsu K, et al.: Glypican-2 binds to midkine: the role of glypican-2 in neuronal cell adhesion and neurite outgrowth. Glycoconj. J. 2001; 18(6): 499–507. Publisher Full Text\n\nMaeda N, Ichihara-Tanaka K, Kimura T, et al.: A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J. Biol. Chem. 1999; 274(18): 12474–12479. Publisher Full Text\n\nRoss-Munro E, Kwa F, Kreiner J, et al.: Midkine: The Who, What, Where, and When of a Promising Neurotrophic Therapy for Perinatal Brain Injury. Front. Neurol. 2020; 11: 568814. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrummond E, Kavanagh T, Pires G, et al.: The amyloid plaque proteome in early onset Alzheimer's disease and Down syndrome. Acta Neuropathol. Commun. 2022; 10(1): 53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson ECB, Carter EK, Dammer EB, et al.: Large-scale deep multi-layer analysis of Alzheimer's disease brain reveals strong proteomic disease-related changes not observed at the RNA level. Nat. Neurosci. 2022; 25(2): 213–225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nGhandi M, Huang FW, Jane-Valbuena J, et al.: Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature. 2019; 569(7757): 503–508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021.\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Characterization Report for Midkine.2021. Publisher Full Text\n\nLaflamme C:Dataset for the Midkine antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "208220", "date": "29 Nov 2023", "name": "Jean-Pierre Bellier", "expertise": [ "Reviewer Expertise Neuroscience", "antibody generation and use." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Ayoubi et al. investigated the specificity of eight commercially available antibodies directed against the human Midkine protein. Midkine is a secreted pleiotropic protein involved in cell proliferation, survival, and the regulation of inflammation. It is often associated with tissue repair and cancer development and has also been shown to be present in amyloid-beta plaques in Alzheimer's disease.\nThe manuscript is well-written, with experiments meticulously executed and technically sound, utilizing HAP1 cell WT and KO as controls. The availability of all datasets on Zenodo is highly commendable and professional.\nThis work reveals that among the antibodies tested, three performed well for Western blotting (WB), and while all showed positive signals for immunoprecipitation (IP), six completely depleted the supernatant of the culture from Midkine protein. Two antibodies performed well in both experiments. Although the authors do not provide a detailed commentary and interpretation of their results, the data presentation is clear enough for readers to identify potentially suitable antibodies for WB and IP studies.\nSpecific comments:\nIt would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells.\n\nFor greater clarity, the authors should specify in the title that their study pertains to human Midkine and mention that HAP1 is a human cell line.\n\nThe term \"high performing\" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion.\n\nIn the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is \"Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]\"\n\nTwo of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this.\n\nHave the authors optimized the working concentration used? If so, they should explain the optimization process.\n\nThe authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility.\n\nFinally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial.\n\nSome references (10-13) are not complete.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "10768", "date": "18 Jan 2024", "name": "Kathleen Southern", "role": "Author Response", "response": "Dear Jean-Pierre Bellier, Thank you for your feedback. We have submitted a modified version of this manuscript, with your requested modifications included. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below. 1. It would have been valuable if the authors had conducted a similar comparison for Immunofluorescence using WT and KO HAP1 cells. We haven’t been able to detect intracellular Midkine by Western Blot. This is because once Midkine is synthesized into a protein, it is quickly secreted. Due to this factor, we expect that detecting intracellular Midkine via Immunofluorescence would be very difficult given the fixation and permeabilization methods used with our standard protocol. 2. For greater clarity the authors should specify in the title that their study pertains to human midkine and mention HAP1 is a human cell line. In the newly submitted version of this article, the title has been updated to indicate that our study pertains to human Midkine. In the results and discussion section, we have clarified that the HAP1 cell line used is human. Thank you for this suggestion. 3.The term \"high performing\" used in the title may be misleading since the study primarily assesses antibody specificity rather than overall performance. If the authors want to use “High-performance” in the title, they should consider providing some interpretation of their data in the discussion. The new title of this article, “A guide to selecting high-performing antibodies for human Midkine for use in Western Blot and immunoprecipitation”. This modification aims to convey that the article’s objective is to guide researchers in selecting high-performing antibodies for their research’s needs. To the introduction, we have included a statement which indicates why the authors chose not to assess the results and draw conclusions on the antibodies performance. We trust these adjustments meet your expectations and prevent any further misinterpretations. 4.In the introduction, the authors mentioned that Midkine (MK) is a potential biomarker and therapeutic target for Alzheimer's disease (AD). The original reference for the work demonstrating MK in senile plaques is \"Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer's disease. Biochem. Biophys. Res. Commun. 192, 246-251. [PMID: 8476427]\" Thank you for providing the original reference for the work done to isolate MDK in senile plaques. It has been included in the new version as reference number 7. 5.Two of the antibodies featured in this study, NBP2-66948 and GTX116089, are discontinued. The authors should mention this. This piece of information has been included in Table 2, where discontinued antibodies are now indicated with a superscript a. 6.Have the authors optimized the working concentration used? If so, they should explain the optimization process. This study for Midkine is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That being said, we have developed universal protocols that has been successful for characterizing antibodies for 85 proteins thus far. In our experiments, a major parameter that we can control to optimize the process is the level of endogeneous protein in the selected cell type. We try to select cell lines, using DepMap portal, with adequate transcript levels of the protein target. 7.The authors should also provide a rationale for selecting the antibodies tested. Many of them are not commonly cited in the literature. An explanation for their choice of antibodies would enhance the manuscript's comprehensibility. The antibodies to be characterization under our standard procedure are donated by YCharOS partners (https://ycharos.com/partners/). These industry partners cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences. 8.Finally, could the authors clarify why it is necessary to deplete the cells? Is it to promote Midkine secretion or to avoid interference from IgG in subsequent experiments? Further clarification on this point would be beneficial. To help us provide a complete response to this comment, could you specify what you mean by “deplete the cells”? 9.Some references (10-13) are not complete. The DOI for citations pertaining to our prior work, available on Zenodo, have been included in the new version submitted. While these references are not found on PubMed, and may seem “incomplete”, they are crucial in helping readers comprehend the protocols used and access the underlying data." } ] } ]	1	https://f1000research.com/articles/12-148
https://f1000research.com/articles/12-367/v1	05 Apr 23	{ "type": "Research Article", "title": "Magnitude and determinants of road traffic accidents in North Gondar Zone; Amhara Region, Ethiopia", "authors": [ "Abay Semegnew Molla" ], "abstract": "Background: Despite efforts that the government has made to minimize the number of casualties or injury levels in Ethiopia, road traffic accidents remain a severe health concern throughout the country and in the study area as well. This study aims to examine the primary contributing variables of road traffic accidents in the North Gondar zone, under 24 administrative.\n\nMethods: The functional class of roads were directly examined on the site to evaluate the existing conditions of the roads which includes the geometric design constraints, functional class, daily traffic, and surfacing materials. Secondary source of data types, previously recorded road traffic accident information was collected from North Gondar zone traffic police departments. Road inventory information was collected from Gondar branch Ethiopian road authority under the specified horizon lines.\n\nResults: The magnitude and frequency of traffic accidents caused by driver-related factors( age, sex, level of education, driving experience, ownership responsibility of a vehicle and driver responsibility, time and day-wise distribution of traffic accidents), vehicle-related factors (failures of mechanical parts and its service age), road related factors( geometric design and construction, inventory information, road surfacing materials), pedestrians, and environmental factors were critically analysed. The result shows that a higher proportion (67%) of traffic accidents are mainly due to driver behaviour related factors than other causative factors. Conclusions: According to the results of the study, bad driving behaviour’s (exceeding the design speed, using a phone while driving, dangerous overtaking, excessive braking, loading above capacities, illegally parking out of public stations should be controlled to reduce the road traffic accident in the study area. In addition, successive up-to-date advanced computerized traffic accident safety training and recording systems should be given at least once a year through regional-based or at a central zone administrative zones for the traffic officers.", "keywords": [ "Road traffic accidents", "causality", "injuries", "property damage", "negative impacts." ], "content": "Introduction\n\nAsegie (2019) indicated that in Sub-Saharan Africa, particularly Ethiopia, where alternative modes of transportation are scarce, road transportation has served as an economic pillar, an inducer of commerce, trade, and traditions, as well as the growth of feelings, cultures, and people's socio-economic lives. This form of transportation has assisted the country's growth by facilitating the movement of agricultural and industrial commodities, as well as services, from one area to the next and to far-flung villages. Worku (2011) noted that road transportation is intended to create a network that connects a variety of infrastructures. Alternative contemporary forms of transportation are too expensive for developing countries such as Ethiopia, so the road transport business is vital. According to Gebrechristos (2014), road transportation has a foundation in the eradication of poverty and offers the country urban-to-urban and urban-to-rural connectivity. The relevance of rural road infrastructure has been recognized in both the country's general development policies and a multitude of sector initiatives (Emmenegger, 2012).\n\nEven if road transportation is a need for subsistence, its traffic accidents are one of the world's most serious problems, including fatalities and property loss. The most prevalent causes of traffic accidents are human factors, vehicle factors, and road conditions (Rolison et al., 2018). Tiruneh et al. (2014) studied that patients who come to Addis Ababa Black Lion Hospital for treatment are more likely to be involved in a road accident, as evidenced by the results of inebriated driving. A six-month institutional-based cross-sectional study on patients at the University of Gondar Comprehensive Teaching and Referral Hospital was conducted to examine road traffic accidents and related factors. Based on the study, results show that there was a high traffic accident among traumatized patients (Honelgn & Wuletaw, 2020).\n\nA community-based cross-sectional research study with a total sample size of 422 people was conducted in Chuko Town. According to the study, poor road conditions were the main cause, accompanied by motorbike overspeeding (Tegegne, 2020). Shamenna et al. (2021) analysed the spatial distribution of road traffic accidents in Hawassa City. As per this investigation, sloppy driving, refusing to secure pedestrians' priority, driving too fast, and failure to give each other space by the drivers are the causes of traffic accidents.\n\nTadege (2020) conducted in Finoteselam town to determine the rate of traffic accidents. In fatal traffic accidents, drivers' lack of driving expertise, inadequate educational level, age of the driver, weekend time driving, and driving in the summer season were listed as important causes of the problem. Using six years of data, Tullu et al. (2013) studied the characteristics of police-reported road traffic accidents in Ethiopia. The researcher observed that there is a higher proportion of pedestrians. The collisions happen during daylight, affect men, and happen in the center of the roads. Rollover on a road tangent, failure to follow pedestrian priority, and overspeeding are all factors that contribute to the traffic accident.\n\nHordofa et al. (2018) cited that road traffic fatalities are seen as a man-made calamity in Ethiopia. The Ethiopian National Road Safety Office claims a fatality rate of 114 per 10,000 cars per year. However, due to an inefficient reporting system, the true figure might be higher. Aside from the loss of life, traffic accidents cost an extra amount of money for medical treatments, physical discomfort, permanent impairment, and travel stress. It also has an impact on patients’ household income, lowering their quality of life and the national economy as well.\n\nAs a result of rapid motorization without adequate regulation, rapid population growth and expansion of the road network, and poor attitudes and safety culture among road users, the likelihood of a road traffic accident is increasing in Ethiopia. Although there are limited activities geared towards combating the problem, they are insufficient by any standard relative to the worsening situation. Road safety management tends not to receive due consideration because of the non-availability of research carried out in the area. Hence, creating a sound road traffic accident database is a very important tool to identify the major causes of the accidents and work towards improvements. In general, without the basis of a dependable traffic accident data recording system, there could be no effective road safety work in our country, specifically in the study area.\n\nRoad accidents and traffic management are issues of vital concern to road authorities. Detecting the risk factors for accidents can contribute to making a road more efficient, safe, and comfortable. This study focuses on issues relating to road surface conditions, traffic facilities, and road users' conduct that lead to traffic accidents. Roads from Gondar town adjacent to Wereda town in North Gondar administrative boundaries were selected to serve as the case study. A field survey of the current traffic accident conditions was conducted on the selected road networks. Previous accidents, road surface defects, and insufficient traffic control facilities that are directly connected to road safety were visually and technically inspected in the analysis. This study relies on the identification of the basic causes of problems along with the selected road networks for effective recommendations and safety management.\n\n\nMethods\n\nThe study was conducted in the North Gondar administrative zone, located in the Amhara region, 745 kilometres north of Addis Ababa. It includes 24 weredas in the zonal study area. The total population was estimated at 2,929,628 (CSA, 2007). This study was a descriptive type of research in which both qualitative and quantitative types of data. It focuses on the assessment and analysis of the road traffic accident causative factors in the North Gondar zone administrative boundaries (the roads bounded in 24 werda’s). Secondary sources of data were used to achieve the intended objective of the research. The main objective of this study was to assess and analyse the major contributing factors to road traffic accidents and their effects on life lost, injuries, and property damage in the specified study area. A five year data which includes road user’s characteristics, vehicle, road and environmental factors in relation to the frequency and intensity of road traffic accidents collected from north Gondar zone police departments and road functional database management systems obtained from Gondar branch Ethiopian road authority are the sources of data for the analysis.\n\nThe five years of police report data (2015 to 2019) for this research study area served as the data sources. Excel (version 2013) and SPSS (version 2020) were used as statistical tools for the data analysis.\n\n\nResults\n\nThere was an incidence of highest records (34 percent and 24.85 percent of personal aggregate effects and property damage) out of the 91 and 507 personal injuries and property damage that occurred in those five years. Furthermore, the age group (19-30 years) had the highest record of person and property damage statistics (56 percent and 62 percent, respectively) than the other age groups.\n\nTable 1 illustrates the driver's age and the types of incidents. Young drivers between the ages of 19 and 30 are the most susceptible, with a total of 598 accidents (56.7 percent) compared to older age drivers (>50 years). It is because younger age groups engage in riskier activities and have less driving experience.\n\nFigure 1 shows that, in addition to age, the educational level has a significant impact on the incidence of road traffic accidents. We believe that better-educated drivers pay more attention and are more concerned about reducing the risk of a road traffic collision than less educated drivers. As a result, a driver's education provides the information, abilities, and attitudes necessary for vehicle safety, both as a driver and as a pedestrian.\n\nAccording to the driving license regulations in Ethiopia, a driver's license can be acquired after completion of junior high school. The highest (29.4 percent) out of 602 aggregate accidents recorded by junior level completed drivers than the others. This indicates that the occurrence of accidents is related to the poor quality of training and the unlawful approval of driving license systems.\n\nFigure 2 illustrates the driving experience and percentage of damages. Driving a vehicle for an extended period and embracing the behaviour of a car helps to minimize the occurrence of road traffic accidents. Inexperience is a flaw that can lead to mistakes in operation.\n\nOut of the 685 cumulative effects of aggregate damage, the highest (87%) of damage was recorded in a driver without owning approved licenses. This indicates that there is a weak control system of driver’s licenses at traffic regulation offices.\n\nFigure 3 shows the number of damages and the responsibilities of the vehicle's owner. The highest (63.2 percent) of 622 road traffic accidents occurred with a hired driver rather than the vehicle's owner. In contrast to the employed driver, the owner causes an average traffic accident of 14.6%. This is the fact that the owner of a commercial vehicle who is using great caution in limiting vehicle speed, increases the likelihood of a traffic collision.\n\nWhen a vehicle collides with other vehicles, a pedestrian, an animal, road curbs, existing buildings, or any immovable barrier such as a tree, or electric pole, a building causes injury, death, and property damage. Trucks are a regular sight on the country's roadways, these vehicles are a vital element of the economy, transporting commodities from one location to another to fulfil the demands of the country's rapidly rising population. Large vehicles, while necessary for a society's existence, can pose a high risk to other small vehicles, especially when truck drivers are careless. Truck accidents of all kinds are responsible for some of the worst roadside destruction. Trucks (small to articulated) and buses (medium to large size) were involved in the highest (39 and 38) percent of the 579 total road traffic accidents shown in Figure 4. This is because truck vehicles are heavier and larger than passenger vehicles, and hence there is a higher risk of a traffic collision.\n\nThe vehicle mechanical issues problems cause road traffic accidents. The issues of vehicle mechanical parts are tire problems, blowouts and skidding due to worn tread, brake issues, slow brake time and brake failure, steering issues, such as pulling or loss of power steering, seatbelts that don't work, brakes that fail, airbags that injure, tires that have defects, or defective accelerators that can be inadvertently triggered. Out of 603 aggregate road traffic accidents recorded the highest (48.3% has come about with any diagnosed mechanical failures than any defined internal and supportive parts of vehicle problems as indicated in Figure 5.\n\nIn parallel to this, the vehicle service age has also an effect on road traffic accidents. In Ethiopia even though the population of motorized vehicles is increased from time to time, it has no approved rule showing the vehicle service age limits which leads to traffic accidents likewise to other developed countries' experiences. Out of 588 aggregate road traffic accidents recorded the highest (29.4% and 27.9%) was committed by vehicle service years of (2-5) years and unknown registered age vehicle groups respectively than any other service age categories as shown in Figure 6. This is due that the country does not well a defined database showing the vehicle service age.\n\nTable 2 shows road inventory information for this study analysis. The table includes the names of road segments, length of the stretches (km), Ethiopian road authority road identification reference, road class, the annual average daily traffic (AADT), the type of surfacing materials, and design code standards of the selected analysis roads, asphalt-covered surfacing material, and DC-4 to DC-6 design code standards, having an average annual AADT of 84 to 1533.\n\nPavement degradation and faults cause sliding, driving off tracks, inappropriate manoeuvring to avoid road imperfections, and extended driver braking distance, which requires immediate care from road and traffic authorities. Besides the type of surfacing materials, dry and wet conditions of the road, poor surfacing, macro, and micro-texture could lead to hydroplaning and inconsistency in tire pavement contact and also a reduction in tire gripping the pavement, which eventually causes accidents. Throughout the study year, 578 (96.2 percent) of the 601 aggregate road traffic accidents occurred on dry asphalt/gravel road surfaces rather than the most common wet soil conditions. As shown in Figure 7, the impacts of this road traffic collision are insignificantly connected to road conditions rather than human causes.\n\nTraffic safety is influenced by the different types of roadways. Although it is difficult to completely avoid traffic collisions, it is feasible to reduce the severity of their consequences by making roads and cars safer and drivers more cautious. Furthermore, by having a better understanding of the influence of different functional classifications of road, the risk may be lowered. Out of the 598 recorded casualties and property losses, in the main access roads with 475 to 1068 annual average daily traffic (AADT) had the highest 275 (46%) than any other functional class, as shown in Figure 8. This indicates that an increase in daily traffic volume above the route's capacity has a negative influence on road traffic accidents.\n\nThe location and road stretch have an adverse effect on road traffic accidents. Figure 9 shows that, of the 527 total accidents recorded, 270 (51.2%) and 118 (22.4%) occurred in rural and residential areas, respectively, compared to other areas. This indicates that in rural areas, the local residents, especially farmers, do not have sufficient awareness and consciousness to protect themselves from road traffic accidents. In contrast, there are many young kids in front of schools who have limited awareness of road traffic accidents, vehicle speeds, sidewalk usage, recognizing pedestrian crosswalks, and other behaviours that might help lower the risk of a road traffic accident.\n\nThrough visual impairments, precipitation, severe winds, and temperature extremes, weather has an influence on driving capabilities, vehicle performance (traction, stability, and maneuverability). As shown in Table 3, the majority of weather-related crashes (547, or 91.9 percent) occur during visible day light (539, or 91.2 percent).\n\nFigure 10 shows the time-wise distribution of road accidents in the study area. It reveals there is a substantial variation in road traffic accidents during different times of the day. Out of 598 total recorded aggregate accidents, the highest (31.6% and 22%) were observed during the time intervals of 8:00 to 12:00 and (15:00 to 17:00) respectively. Hence, the highest amount of traffic flow was observed in the daytime rather than in the night, since a higher percentage of traffic accidents were recorded.\n\nAs shown in Figure 11, there was less (2%) fluctuation among the days (Monday to Saturday) than there was on Sunday among the 597 traffic accidents registered in the police department. There have been fewer (11.2 percent) road traffic accident recordings since this date. This shows that there is less commercial vehicle traffic flow and passenger movement on Sundays, and so fewer accidents were seen.\n\nThe incidence of road traffic injuries was 89 (38.7%) of the total of 230 aggregate damage that occurred. The majority of these victims were farmers, which accounted for 114 (49.6%), followed by students, which constituted 53 (23%) more victims than any other category of people as indicated in Figure 12.\n\n\nDiscussions\n\nIn Ethiopia, including the study area, the basic accident data itself is collected by only the police and is recorded in a notebook using a locally developed standard format. In most cases, this takes place as a result of attending a road accident, but there are occasions when the accident is reported after the event at a police station. In this case, the missing of valuable information about the traffic accident's causative factors leads to misinterpretation of recommended actions to be taken. So, there should be a formal, centrally developed traffic accident recording standard in Ethiopia that may be adopted from developed countries having their systems automated. This will provide valuable information for academic issues and research.\n\nIn place of manual recording traditions, there should be an automated computerized web-based database recording system that works through the country regions also applicable in the study area, which will enable the traffic police offices and other road safety stakeholders to get summarized online traffic accident information at various levels easily and quickly. This can be done cooperatively with the federal police commission's central traffic accident analysis department and global consulting agencies that have good exposure to traffic database management systems. The system may include different subsystems for administrative and accident registration to meet its core functionalities, which are managed in different local languages.\n\nAs per the investigation, the cooperation between the concerned sectors which includes the road transport, traffic police departments, and the Ethiopian road authority and respective construction and housing development offices is low even though the traffic accident impact is high. In this case, a uniform centralized rule should be dispatched from the Amhara regional bureau to avoid the disparities interaction in this sector for traffic accident reduction.\n\nA Higher proportion (67%) of traffic accidents are mainly due to driver behaviour-related factors than other causative factors. In this case, bad driving behaviours such as tailgating (driving too closely behind another car), exceeding the design speed, using a phone while driving, failing to indicate when turning directions, dangerous overtaking, excessive braking, a lack of understanding of roundabouts, loading and seating above the specified loading capacities and number of chairs, illegally parking and loading out of public stations.\n\nA successive traffic accident safety trainings supported by up-to-date advanced computerized control and recording systems will be given at least once a year through regional-based or at a central zone administrative zones for the concerned traffic officers.\n\nThe traffic police officers should take higher responsibility to reduce the traffic accidents impacts in the area. If possible, it is better to stop an illegal communication with commercial vehicle drivers for the sake of their personal business enhancement. They should also give priority to passenger’s safety and worry about to human life lost. This should be managed through scheduled panel discussions with traffic officers and annual rewards.\n\nCurrently in the study area, annual traffic accident day celebrations, student traffic police formation and trainings, and pedestrian side keeping are practiced, and this should be promoted and continued further to increase traffic accident safety. In general, every individual, private sector government, and non-governmental organization should collaborate and play their respective roles to achieve an effective zero percent valuation of road traffic accidents in this area.\n\n\nConclusions\n\nEthiopia, including the study area, is in the midst of a major road safety crisis. Thousands of people who work on the roads are slain every year. The prevalence of road traffic accidents has increased even though the government has given special attention to reducing its effect.\n\nA descriptive type of research, in which both qualitative and quantitative types of data from secondary sources were used, was collected from previous traffic police reports. This kind of data collection was made by collecting the previous five years police report road traffic accident (2015–2019) in North Gondar zone police departments and functional road information from Gondar branch Ethiopian road authority offices.\n\nThe study focuses on analysing the frequency of traffic accidents caused by driver-related factors including age, sex, level of education, driving experience, ownership responsibility of a vehicle and driver responsibility, time and day-wise distribution of traffic accidents, vehicle-related factors like failures of mechanical parts of the vehicle and its service age, road geometric design and construction considerations like road inventory information, road surfacing materials, pedestrians, and environmental factors.\n\n\nData availability\n\nZenodo. Magnitude and determinants of road traffic accidents in North Gondar Zone, Amhara Region, Ethiopia. DOI: https://doi.org/10.5281/zenodo.6948625 (Molla, 2022).\n\nThis project contains the following underlying data:\n\n- Number and types of a road traffic accidents in relation to road and road user, environmental and time related and vehicle related factors\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).", "appendix": "Acknowledgments\n\nI acknowledge the support of the North Gondar traffic police department, Central Gondar road and transport office, Gondar branch Ethiopian road authority in the collection of traffic police reports data and road functional information for the analysis and presentation of the study.\n\n\nReferences\n\nAsegie Y: Contributing Factors of Traffic Accident and Their Possible Countermeasures in Debre Berhan Town, Ethiopia. American Journal of Civil Engineering and Architecture. 2019; 6(February): 0–6. Publisher Full Text\n\nCSA: 2007 population and housing census of Ethiopia administrative report central statistical authority Addis Ababa (issue April).2007.\n\nEmmenegger R: The Roads of Decentralisation The History of Rural Road.2012.\n\nGebrechristos N: Ethiopian Practices in Urban-Urban and Urban-Rural Linkages from Road Transportation Perspectives in Hawassa City, Ethiopia. Economics and Sustainable Development. 2014; 5(27): 227–236.\n\nHonelgn A, Wuletaw T: Road traffic accident and associated factors among traumatized patients at the emergency department of University of Gondar Comprehensive Teaching and Referral Hospital.2020; 4(9).\n\nHordofa GG, Assegid S, Girma A, et al.: Prevalence of fatality and associated factors of road traffic accidents among victims reported to Burayu town police stations, between 2010 and 2015, Ethiopia. Journal of Transport & Health. 2018; 10(November 2017): 186–193. Publisher Full Text\n\nMolla AS: Magnitude and determinants of road traffic accidents in North Gondar Zone, Amhara Region, Ethiopia [Data set]. In f1000research. Zenodo. 2022. Publisher Full Text\n\nRolison JJ, Regev S, Moutari S: What are the factors that contribute to road accidents? An assessment of law enforcement views, ordinary drivers’ opinions, and road accident records. Accident Analysis & Prevention. 2018; 115(August 2017): 11–24. Publisher Full Text\n\nShamenna A, Senbeta BA, Bezabih AA: Spatial Distribution of Road Traffic Accident at Hawassa City Administration, Ethiopia. Ethiopian Journal of Health Sciences. 2021; 31(4): 793–806. Publisher Full Text\n\nTadege M: Determinants of fatal car accident risk in Finote Selam town, Northwest Ethiopia. BMC Public Health. 2020; 20(1): 624. PubMed Abstract \| Publisher Full Text\n\nTegegne KT: Prevalence of Road Traffic Accidents and Associated Factors in Chuko Town.2020; 9(2): 76–84.\n\nTiruneh BT, Dachew BA, Bifftu BB: Incidence of Road Traffic Injury and Associated Factors among Patients Visiting the Emergency Department of Tikur Anbessa Specialized Teaching Hospital, Addis Ababa, Ethiopia. Emerg. Med. Int. 2014; 2014: 1–6. PubMed Abstract \| Publisher Full Text\n\nWorku I: Growth in Ethiopia 1. Ethiopian Journal of Economics. 2011; 19(October): 101–146.Reference Source" }	[ { "id": "192799", "date": "14 Aug 2023", "name": "Svetlana Cociu", "expertise": [ "Reviewer Expertise Injury", "road traffic injuries", "traumatic brain injury", "health promotion", "global health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reflect a very informant issue. Results are significant for the scientific environment. I would recommend a slight modification:\n\nIn the abstract, the result section needs to refer to the results obtained and all the variables used need to be replaced in the methodology part. The methodology is not completed, it would be interesting more details about how data were obtained and what statistical tools were used. The result part is well organized - I suggest revising the figures and in some of them - to do average of the study period. The discussion part needs revision - data comparable with other studies should be given. The conclusion needs to refer to some solutions based on the obtained results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10432", "date": "29 Nov 2023", "name": "Abay Semegnew Molla", "role": "Author Response", "response": "Thank you very much for your well informed comments." } ] }, { "id": "192802", "date": "31 Oct 2023", "name": "Puspa Raj Pant", "expertise": [ "Reviewer Expertise Road Traffic Injury Prevention", "Injury Prevention" ], "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\nAuthor could have referred to some global literature to give an idea about recognised risk factors and effective measures to address them. Some of the paragraphs in introduction section look more appropriate to be mentioned in the Discussion.\nThe objective mentioned in Abstract, Introduction, and Discussion section is not consistent. The design of the primary study conducted on road surfaces in North Gondar area is not well described. Although \"Qualitative\" design is mentioned, no results or discussion is seen in the manuscript.\n\nThe Methods section needs clear details on Quantitative, Qualitative and Secondary data analysis. Currently the data analysis is very thin. The author could have generated some crosstabulations.\nThe manuscript has not made justice to identify the \"determinants\" as mentioned in the Title.\n\nThe first three paragraphs of Discussion look more like recommendations. The author could compare and contrast their findings (as some examples mentioned in introduction section).\nConclusion looks like a summary of methods.\nReferences need to be cited in full in the Bibliography: Tegegne KT; Honelgn A; Wuletaw T; Emmenegger R.\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "212327", "date": "31 Oct 2023", "name": "Mireia Faus", "expertise": [ "Reviewer Expertise Road traffic", "safety", "communication", "public health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper analyzes the main variables contributing to road accidents in a region of Ethiopia. The causes and variables related to road accidents are a widely researched topic internationally. However, the study in a specific region of Ethiopia may have important practical applications at the local level, as well as for regions or countries with similar socio-demographic characteristics.\nIn the following, I will make some recommendations that I consider can improve the quality of the paper.\nI recommend further developing the introduction, including the main factors involved in road accidents, such as the human factor, the infrastructure factor and the vehicle factor. In this sense, two subsections could be included, referring to 1) Literature review, synthesizing the main research on the subject, especially those carried out in emerging countries, and 2) The case of Ethiopia, explaining the country's situation with respect to road accidents and citizens' mobility.\nThe methodology should describe the variables evaluated, the type of statistical analysis carried out, the ethical aspects of the study, etc.\nThe data are merely descriptive. Therefore, the discussion cannot be a mere summary of the data obtained. This section should explain and contrast the data obtained with other research in this field of study. Thus, the authors should answer questions such as: were the results in accordance with expectations, are the results congruent with other similar research? And, if not, what elements explain the discrepancies that have occurred? The limitations of the study should also be included in a specific section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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? No", "responses": [] }, { "id": "192796", "date": "31 Oct 2023", "name": "Aliyu Mustapha", "expertise": [ "Reviewer Expertise Road Traffic Accidents", "Automobile Technology", "Technical Vocational Education and Training", "Educational Technology", "ICT in Education", "ICT for Education", "Road Signs and Ergonomic" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStrengths\nRelevance of the Topic: The article addresses a highly relevant and significant issue – road traffic accidents, which are a major public health concern not only in Ethiopia but globally.\n\nClear Objective: The article clearly states its objective, which is to examine the primary contributing variables to road traffic accidents in a specific region, the North Gondar Zone. This clear focus helps readers understand the study's purpose.\n\nComprehensive Data: The study uses a five-year dataset, which provides a substantial amount of data to analyze the factors contributing to road traffic accidents. This strengthens the validity of the findings.\n\nRecommendations: The article provides practical recommendations for reducing road traffic accidents, specifically focusing on controlling bad driving behaviors and implementing advanced computerized traffic accident safety training and recording systems. These recommendations have the potential to impact policy and safety practices.\n\nStructured Layout: The article follows a logical structure, with clear sections for the introduction, methods, results, discussion, and conclusion. This organization makes it easy for readers to follow the research's progression.\n\nUse of References: The article effectively references prior research to provide context and support the need for this study.\n\nClear Recommendations: The article provides clear and practical recommendations for improving the road safety situation in Ethiopia and the study area. Suggestions for the implementation of an automated, web-based traffic accident recording system and the need for collaboration among various sectors are valuable.\n\nEmphasis on Driver Behavior: The article appropriately highlights the importance of driver behavior as a significant contributor to road traffic accidents, and it provides specific examples of bad driving behaviors. This focus on behavior addresses a critical aspect of road safety.\n\nEmphasis on Collaboration: The emphasis on improving cooperation between different sectors, including road transport, traffic police, and the Ethiopian road authority, is a valuable point for addressing road safety comprehensively.\n\nDiscussion of Ongoing Practices: The article highlights existing practices such as annual traffic accident day celebrations, student traffic police formation, and training, which should be promoted to enhance traffic accident safety. These practices are positive and should be continued.\nWeaknesses\nData Collection Details: The article lacks specific details about the data collection and analysis methods used. Readers may want more information on how data was gathered, processed, and analyzed to ensure the validity and reliability of the results.\n\nLiterature Review Depth: While the article references existing literature, it could benefit from a more comprehensive literature review that delves deeper into global and regional trends in road traffic accidents. This would provide a broader context for the specific findings in the North Gondar Zone.\n\nLimited Discussion Implications: The discussion section could be expanded to explore the broader implications of the findings. How might these findings impact road safety policies, practices, or interventions not only in the North Gondar Zone but also in other regions? Providing a more extensive discussion could enhance the article's impact.\n\nClarity in Language: While the article is generally clear, there are areas where the language could be refined for greater clarity and precision. Some sentences are complex and might benefit from simplification.\n\nLack of Data: The article does not present any new data or statistical analysis related to road traffic accidents in the North Gondar Zone. It mainly consists of recommendations and general statements about the problem without supporting data or analysis.\n\nRepetitive Statements: The article repeats certain points, such as the importance of an automated recording system and the role of driver behavior, without providing substantial additional insights or examples.\n\nAbsence of Literature Review: The article does not reference existing literature or research on road safety in Ethiopia or the North Gondar Zone. Including a literature review would provide context for the recommendations and reinforce the importance of the issue.\n\nIt is not always good to start a paragraph with citation, always start a paragraph with your own words, then support with citation(s). “Check the first, fourth and fifth paragraphs of the introduction”.\n\nThe first-two paragraphs of the conclusion are “Summary” not “Conclusion”. Re-write the conclusion based on your objectives.\n\nData analysis used is mainly descriptive. You should add inferential statistics to make educated guesses or inferences about populations based on data from a sample. Formulate hypotheses based on your research questions.\n\nProvide a subheading for recommendations and limitation(s) based on your research objectives\nConclusion In conclusion, the article addresses a crucial issue and provides valuable recommendations to mitigate road traffic accidents. Its strengths lie in its relevance, clear objectives, comprehensive data, and practical recommendations. However, there is room for improvement in terms of providing more details about data collection methods, expanding the literature review, and further discussing the broader implications of the research 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? 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/12-367
https://f1000research.com/articles/13-855/v1	31 Jul 24	{ "type": "Case Report", "title": "Case Report: Multidisciplinary management of severe blast-related craniofacial and ocular injuries at artisanal gold mining sites in Niger: An unusual case report and comprehensive literature review", "authors": [ "Ousmane Issoufou Hamma", "Yahouza Boka Tounga", "Haboubacar Abdou Moussa", "Tidjani Mahamat Hissene", "Ibrahim Moussa Daouda", "Fassouma Laouali Abba", "Kpègnon Nicaise Agada", "Garba Ide", "Assoumane Issa Ibrahim", "Inoussa Daouda Bako", "Aminath Bariath Kelani", "Maman Sani Chaibou", "Rachid Sani", "Yahouza Boka Tounga", "Haboubacar Abdou Moussa", "Tidjani Mahamat Hissene", "Ibrahim Moussa Daouda", "Fassouma Laouali Abba", "Kpègnon Nicaise Agada", "Garba Ide", "Assoumane Issa Ibrahim", "Inoussa Daouda Bako", "Aminath Bariath Kelani", "Maman Sani Chaibou", "Rachid Sani" ], "abstract": "Blast-related craniofacial and ocular injuries have traditionally been associated with battlefields. In civilian populations, these injuries were infrequent, primarily arising from accidental explosions or terrorism incidents. Explosions can inflict severe damage to the neurological, ocular, and auditory systems through blast waves, high-velocity foreign bodies, and thermal radiation. The pathophysiology of blast-related craniofacial injuries is specific and complex, with severe cases often involving both penetrating and blunt trauma, leading to high mortality and morbidity rates. These injuries necessitate multidisciplinary management and can be defined as intracranial polytrauma. Recently, Niger has seen a surge in blast injuries, predominantly due to the increase in clandestine artisanal gold mining. Managing these injuries in resource-limited settings poses significant challenge. Comprehensive national data on these injuries are sparse due to high pre-hospital mortality rates and their infrequent occurrence, resulting in limited experience among our local physicians in their management. We present a rare case involving an artisanal gold miner with a history of smoking and previous concussions from explosion exposures. The patient was transferred to our hospital following severe craniofacial and ocular injuries caused by an accidental dynamite explosion at a mining site. On admission, the patient presented altered consciousness, agitation, unstable vital signs, multiple craniofacial wounds, including a large frontal wound with brain substance extrusion, diffuse facial burns, left globe rupture, and rhinorrhagia. After resuscitation and stabilization, brain imaging revealed multiple complex craniofacial fractures with foreign bodies. The patient underwent multidisciplinary surgical management. However, the postoperative course was complicated by post-concussive syndrome, and infection of the surgical wounds, necessitating surgical revision. Following the second surgery, the postoperative course was uneventful, although the patient experienced reduced visual acuity. This case highlights the management challenges in Niger and underscores the urgent need for clinical studies and training for gold miners to enhance safety practices in their activities.", "keywords": [ "Blast injury", "blast-related craniofacial trauma", "blast-induced neurotrauma", "blast-related ocular trauma", "developing countries", "case report" ], "content": "Introduction\n\nIn recent years, blast-related craniofacial and ocular injuries have increasingly occurred outside of battlefields, becoming a significant concern in civilian settings.1–3 The pathophysiology of these injuries is distinct due to the interaction of blast waves and thermal radiation with biological tissues, which exacerbates both penetrating and blunt injuries.2,4,5 Blast waves cause rapid changes in the pressure within the brain, ocular globe, and middle ear, leading to tension and shearing forces that can result in contusions, intracranial hemorrhage, vitreous hemorrhage, globe rupture, retinal detachment, tympanic membrane perforation, etc.2,6 Consequently, severe blast-related craniofacial injuries can be defined as intracranial polytrauma, necessitating immediate multidisciplinary management.\n\nThe medical community’s interest in civilian blast-related craniofacial injuries has grown due to the rise in terrorism (improvised explosive devices), accidental explosions, and civilian casualties from military conflicts (heavy munitions firing).4,5,7 In our country, the primary causes of these injuries are explosion accidents and terrorism, particularly accidental dynamite explosions at artisanal gold mining sites. However, comprehensive national data on these injuries are sparse due to high pre-hospital mortality rates and their rarity, resulting in limited experience among our physicians in managing such cases.\n\nOur objective is to report a rare and well-documented case of blast-related severe craniofacial and ocular injuries at an artisanal gold mining sites. This report aims to highlight the management challenges in developing countries like Niger, emphasize the urgent need for clinical studies on blast-related craniofacial trauma in resource-limited settings, and advocate for safety training for gold miners.\n\nThis case report has been reported in line with the SCARE Criteria.8\n\n\nCase report\n\nA 25-year-old married male, an illegal artisanal gold miner, with a history of smoking and two prior concussions from explosions exposure (without any apparent serious injury), was referred to our hospital by a peripheral health center sustaining severe craniofacial injuries from a dynamite explosion. The accident occurred three hours prior, while he was installing dynamite accidentally it a gold shaft, resulting in multiple deaths. The patient, found comatose with multiple sharp missile craniofacial and ocular injuries and diffuse facial burns, was the sole survivor and was transported by a personal vehicle to a health center 120 km away. There, he was resuscitated and stabilized with intravenous crystalloid fluids (0.9% sodium chloride saline solution), analgesia (paracetamol 1 g), and antibiotic prophylaxis (ceftriaxone 2 g). He was then transferred by ambulance to our hospital’s emergency department without intubation.\n\nOn admission, the patient was agitated with an altered state of consciousness (Glasgow Coma Scale [GCS] 9/15: Y1 V3 M5) and hemodynamically and respiratory unstable, with a blood pressure of 79/44 mmHg, pulse rate of 156 beats/min, respiratory rate of 24 breaths/min, and oxygen saturation 86% on 10 L/min oxygen via a non-rebreathing mask. He was sedated with midazolam at 4 mg/h via electric syringe pumps. Oro-tracheal intubation and administration of crystalloid fluids and emergency drug (Adrenaline 0.75 mg) stabilized his vital parameters to a blood pressure of 144/64 mmHg, pulse rate of 105 beats/min, respiratory rate of 14 breaths/min, and oxygen saturation 96%. His core temperature was 37-37.5°C, and his body mass index (BMI) was 20.75 kg/m2. Craniofacial examination revealed multiple wounds, including a large penetrating left frontal wound with brain substance extrusion, a penetrating left maxillary sinus injury, diffuse facial burns, and moderate rhinorrhagia (Figure 1). Ophthalmological examination showed laceration of the left eyelids, globe rupture with disorganization of the ocular structures, and a complex fracture of the left orbital frame. The right eye had corneal edema and intracorneal foreign bodies. Examinations of the cervical spine, thorax, abdomen, limbs, and motor function were normal.\n\n(A-D) Large penetrating left frontal wound with brain substance extrusion (green arrows), laceration of the left eyelids, globe rupture with disorganization of the ocular structures (blue arrows), penetrating the left maxillary sinus injury (white arrow), rhinorrhagia (red arrows), and diffuse facial burns from the explosion.\n\nStabilization was continued following the ATLS (Advanced Trauma Life Support) guidelines, including intravenous crystalloid fluids, multimodal analgesia (paracetamol 6 g/24 h, tramadol hydrochloride 400 mg/24 h), 96-hour prophylactic antibiotic (ceftriaxone 4 g/24 h), and nasal packing. Blast-related severe craniofacial and ocular injuries were diagnosed. Non-contrast brain computed tomography (CT) revealed a large left frontal fracture with intraparenchymal bone fragments and foreign bodies, a left orbital frame fracture with intraorbital foreign bodies, maxillary sinus fractures with foreign bodies, and a skull base fracture associated with a fracture of the left orbital inner wall (Figure 2). Laboratory tests showed an inflammatory process with hyperleukocytosis (17,950 cells/μL, reference: 4,000 – 10,000 cells/μL), granulocytosis (80.04%, reference: 50-70%), elevated C-reactive protein (CRP) (24 mg/L, reference: < 6 mg/L), and anemia (10.2 g/dL, reference range: 11.0-16.0 g/dL). Renal and hepatic function tests were normal, with no ionic or coagulation disorders. A whole blood transfusion was administered, and prophylactic antiepileptic (phenobarbital 100 mg/24 h) was systematically instituted.\n\n(A-F) Large left frontal fracture with intraparenchymal bone fragments and foreign bodies (red arrows), fracture of the left orbital frame with intraorbital foreign bodies (blue arrows), fracture of the left maxillary sinus with foreign bodies (white arrows), and skull base fracture associated with a fracture of the left orbital inner wall (green arrows).\n\nAfter informing the patient’s family of the poor prognosis, multidisciplinary surgery involving neurosurgeons, ophthalmologists, and a maxillofacial surgeon was performed under general anaesthesia with the patient in the spine position. Initially, the right eye was lavaged with saline, and superficial intracorneal foreign bodies were removed (Figure 3A). Subsequently, a large penetrating frontal injury was approached via a bicoronal incision; limited surgical debridement was performed, followed by a watertight duroplasty, cauterization and closure of the frontal sinus (Figure 3B, 3C). Enucleation of the left eye, and surgical care of maxillary sinus injury were performed. Postoperative care in the general intensive care department included intubation, crystalloid fluids, multimodal analgesia, intravenous antibiotic prophylaxis, sedation, local antibiotic prophylaxis for right eye with ciprofloxacin 0.3% (eye drops and ointment) four times daily, alternating with artificial tears. On the third day, the patient was transferred to neurosurgery department.\n\n(A) Large penetrating left frontal wound with brain substance extrusion (green arrow), laceration of the left eyelids and globe rupture with disorganization of the ocular structures (blue arrow), right eye lavage with saline, and removal of superficial intracorneal foreign bodies (red arrow);\n\n(B) Lost-bone craniotomy showing a large dural breach with loss of left frontal lobe substance;\n\n(C) Watertight duroplasty after limited surgical debridement (green arrow), complex fracture of the left orbital roof communicating between the orbit and skull base (blue arrow);\n\n(D) Removal of granite fragments from craniofacial injuries (white arrows).\n\nDuring the first postoperative week, sedation continued due to agitation, and recovery of clear consciousness was slow. Phenobarbital was replaced with sodium valproate 1500 mg/24 h for two weeks. On the third and fifth days, examination of the surgical sites revealed necrosis then dehiscence and infection of surgical wounds (skin around the frontal surgical wound, and the left eyelids) (Figure 4). Biological control tests showed an accelerated erythrocyte sedimentation rate (ESR) of 43 mm/hour and elevated CRP (58 mg/L, reference: < 6 mg/L), prompting intravenous probabilistic antibiotic therapy (amoxicillin/clavulanic acid 3g/187.5 mg/24 h, metronidazole 1500 mg/24 h). A surgical revision was performed on the seventh day, and greasy dressing was applied postoperatively until the surgical wounds healed. By the second week, the patient became less agitated and regained clear consciousness but experienced post-concussive syndrome with headaches, fatigue, insomnia, and concentration difficulties. Psychiatric evaluation recommended conservative treatment. The first ophthalmological examination after regaining clear consciousness revealed light perception. The third-week check-up was good, except for the electroencephalogram (EEG) which showed epileptic activity, necessitating a six-month antiepileptic treatment. All antibiotics (intravenous and local) were discontinued on the 21st day of hospitalisation. Ophthalmological control examination showed corneal clearing and improved visual acuity (6/10). However, the frontal surgical wound showed significant delay in healing, despite good granulation during the first week after surgical revision (Figure 5A). Discharge was authorized on the 40th day of hospitalization, with the surgical wounds were almost healed (Figure 5B). A brain CT with contrast prior to discharge showed good evolution (Figure 6). The patient continued with fat dressings at a nearby peripheral center and regularly consulted our hospital. Post-concussive syndrome symptoms decreased significantly by the second month, with residual headaches and slight fatigue disappearing by the third month. He resumed normal activities in the fourth month, as the symptoms had disappeared and the wounds had completely healed. A follow-up EEG at six months showed spike waves, leading to another six-month renewal of antiepileptic treatment. An eighth-month brain CT with contrast showed good progress but indicated a forming porencephalic cavity (Figure 7). Cranioplasty was planned for the ninth postoperative month. Unfortunately, the patient later died in a gold mine collapse.\n\n(A) On third postoperative day, desiccation of the frontal surgical wound (green arrow), and early dehiscence of the left eyelid (blue arrow);\n\n(B) On fifth postoperative day postoperative, early necrosis and dehiscence of the frontal surgical wound (green arrow), and necrosis of the left eyelid (blue arrow);\n\n(C and D) On seventh day postoperative, advanced necrosis and infection of the frontal surgical wound (green arrows), and the left eyelid (blue arrows).\n\n(A) On ninth day post-surgical revision, good granulation of the frontal surgical wound (green arrow), and the left eyelid (blue arrow);\n\n(B) On thirty-second day post-surgical revision, advanced healing of the frontal surgical wound (green arrow) and the left eyelid (blue arrow).\n\n(A-F) Evolution of contusion and loss left frontal lobe substance forming fronto-polar hypodensity (red arrows), and remodeling of the orbital cavity after enucleation (blue arrows).\n\n(A-D) Formation of a porencephalic cavity (red arrows), and remodeling of the orbital cavity after enucleation (blue arrows).\n\n\nDiscussion\n\nBlast-related civilian craniofacial and ocular injuries in Niger predominantly arise from terrorism and accidental explosions at artisanal gold mining sites. Globally, such injuries in general civilian population is more commonly linked to fireworks, firecrackers, mine gas, detonators, and containers.9,6 In Niger, the high pre-hospital mortality rate and lack of consultation for mild cases result in under-diagnosis and incomplete national data, resulting in limited experience among our physicians in managing such cases. Explosions are sudden and devastating, often causing numerous casualties and significant morbidity among survivors.6 According to Zhong et al.,6 limb injuries are the most common complication of systemic explosion damage in the general civilian population, followed by craniocerebral injuries. Our patient sustained only craniofacial injuries, with the rest of his body spared, and could not recall his position during the explosion. Dynamite explosions can cause a variety of traumatic injuries due to the blast wave, foreign bodies propelled by the explosions, and released heat, with injuries generally categorized as primary to quaternary.5,7,10 Our patient was the sole survivor of the accident, presented all possible categories of craniofacial lesions (penetrating, blunt injuries, blast injuries), resulting a high morbidity state. Unfortunately, the other gold miners sustained fatal injuries, and we do not know the proportion of craniofacial injuries compared to other injuries (limbs, thorax, abdomen, etc.).\n\nBlast-related craniocerebral trauma involves multiple pathophysiological mechanisms, including blast wave transmission through the cranium, skull flexure, thoracic mechanism, translation and rotation head acceleration, and cerebrospinal fluid (CSF) cavitation.11 During blast wave transmission through the cranium, shock waves travel almost unimpeded through nerve tissue, its envelopes, globe, and ear, resulting in a rapid pressure peak followed by negative pressurization. This leads to the compression and expansion of the brain, globe, and tympanic membrane in rapid succession.4,12 Changes in pressurization caused by shock waves can result in tension and shearing of brain tissue, blood vessels, and globe, as well as cavitation of CSF. These changes can cause hemorrhagic contusions, intracranial hemorrhages due to alterations in blood-brain barrier (BBB) permeability, diffuse axonal damage, retinal detachment, tympanic membrane perforation, etc.1,2,5,11,13 Additionally, neuroinflammation, vasospasm, neural loss, oxidative stress, and metabolic changes are observed in moderate and severe trauma, with the most significant changes noted in the frontal cortex and basal ganglia.11 Patient preoperative assessment of the lesions revealed mainly fractures and hemorrhagic contusions, primarily caused by the penetration of high-velocity fragments rather than blast waves. Cerebral edema, intracranial hemorrhages, and diffuse axonal damage were minimal.\n\nThe ocular globe is particularly vulnerable to blast trauma due to its exposure, incompressibility, fluid content, rich vascular network, fragile tissues, and fine structures. Blast-related ocular injuries can lead to impaired visual acuity and even blindness.6,9,13 Ocular damage is almost inevitable in cases of blast-related civilian craniofacial trauma, especially since illegal gold miners do not wear personal protective equipment, hence our patient’s bilateral ophthalmic damage. Over the past decade, ocular injuries have been the leading cause of vision loss globally, affecting developed, developing, and high-income countries alike.9 Unfortunately, the incidence of civilian blast injuries is increasing in our region, as the low socio-economic status of the rural population has led to gold rushes.14 Gold mining activities are predominantly carried out by young adults, and life at clandestine gold mining sites is precarious, exposing miners to numerous diseases (infectious, environmental, etc.) and blast injuries. This observation aligns with scientific literature, indicating that blast injuries mainly affect young adults.1,5,7,13,15 The remoteness and inaccessibility of certain illegal gold mining sites makes pre-hospital treatment of injured individuals extremely challenging. Patients are transferred in personal vehicles or poorly equipped ambulances, resulting in unstable conditions on admission.\n\nPatients with severe craniofacial and ocular injuries from explosions requires comprehensive management, involving a multimodal approach that incorporates data from neuroimaging (magnetic resonance imaging [MRI], functional MRI [fMRI], diffusion tensor imaging [DTI], positron emission tomography [TEP], magnetoencephalography, and electroencephalography), biomarkers (p-tau, glial fibrillary acidic protein, etc.), and neurobehavioral assessments.2,5,16 Certain abnormalities linked to neuroinflammation and oedema caused by BBB damage and oxidative stress have been identified as the causes of neuropsychiatric symptoms, but are very difficult to detect with basic imaging (brain CT, MRI). Detection of these abnormalities in the acute phase is crucial for diagnosis, prognosis and patient follow-up, requiring advanced neuroimaging techniques such as fMRI, TEP, and DTI.2,16 Nevertheless, sequelae lesions are more easily detected in the late phase, even on basic imaging. However, access to brain imaging is limited in Niger due to the absence of many advanced neuroimaging techniques, restricted accessibility for basic imaging, and the financial difficulties patients face in obtaining examinations. Our patient was only able to undergo a preoperative brain CT and faced difficulties obtaining two postoperative brain CT contrast injections.\n\nBlast-related severe craniofacial and ocular injuries necessitate immediate multidisciplinary management and can be defined as intracranial polytrauma. A psychiatric evaluation should be included in their management regardless of the trauma’s severity, as patients with blast-related head trauma are likely to develop various neuropsychiatric disorders (behavioral deficits, post-concussion syndrome, post-traumatic stress disorder, chronic traumatic encephalopathy, etc.) within 2-4 weeks.17,18 Our patient developed post-concussion syndrome during the second week of postoperative follow-up, which was managed conservatively. However, with his history of multiple blast exposures, he is also at risk of developing chronic traumatic encephalopathy.16,19 During follow-up, some patients with neuropsychiatric disorders also show certain neuroimaging signs, notably the loss of white matter integrity and alterations in cortical volume and thickness.11 Unfortunately, we were unable to follow the patient long enough to see the sequellar lesions on the brain CT, due to his accidental death on the gold mining site after resuming his activities. He had also presented with globe rupture and the presence of intraocular foreign bodies only in the left eye, while the right eye was relatively spared by the explosion. Fortunately, the right eye showed no signs of poor prognosis, such as globe rupture or perforation, retinal detachment, or the presence of intraocular foreign bodies.9 Persistent poor postoperative visual acuity in the right eye would have led to severe visual handicap or even total work incapacity for our patient. Despite our limited resources, our patient has a relatively good postoperative outcome, as he had fewer severity factors, including an initial GCS > 9/15 and the absence of tympanic membrane perforation.7\n\nArtisanal gold panning in our country causes numerous health problems, particularly among young adult miners. These problems may require frequent medical attention, entail high financial costs, and result in loss of working days.14,15 Health issues observed on artisanal gold mining sites include disabling after-effects (visual impairment, motor impairment, etc.) resulting from severe head trauma (accidental dynamite explosions, falls from great heights, etc.) and chronic disabling illnesses (chronic pneumocardiopathy, mental disorders, etc.) due to prolonged exposure to chemicals, dust, alcohol and tobacco.15 Moreover, clandestine mining is one of the most dangerous working environments. According to the International Labour Organization, the accident rate is six to seven times higher than in industrial mining, particularly during the excavation stage.15,20 Many adult young artisanal gold miners, like our patient, have suffered severe, lifelong visual impairments as a result of accidents. Some injuries were severe enough to impair their ability to work, while others rapidly progressed to total work incapacity, becoming a burden to their families. Unfortunately, in Niger, the working population (aged 15-64) represented 47.8% of the population in 2022, making it difficult to manage the consequences of these associated health problems, and potentially placing a burden on the healthcare system and the economy.21 However, artisanal gold panning has developed over the years since the first gold rush in Koma Bangou, and it is now a source of income for hundreds of thousands of unemployed people.14 For our patient, clandestine artisanal gold panning is the only activity in his locality that enables him to provide a decent living for his family and relatives. Niger does not have the necessary means to implement coercive measures to put an end to clandestine gold panning due to the vastness of the territory to be controlled and the difficult access to certain areas. It is, therefore necessary to implement a participative management approach involving public decision-makers, the local population, and the gold miners to better regulate and control gold panning activities. Additionally, it is crucial to make gold miners aware of the dangers of some of their mining tools and methods (explosives, mercury, cyanide, etc.) through health and safety training. This training should encourage them to purchase personal protective equipment, such as helmets and goggles, as well as quality machinery. It is also essential to provide isolated gold mining sites with health support and pre-hospital care facilities.\n\nOur study has several limitations. Firstly, the small number of cases and the absence of multimodal neuroimaging did not allow us to obtain comprehensive data on craniofacial trauma caused by accidental explosions on gold mining sites, and to identify aggravating factors. Secondly, the lack of prolonged follow-up (several years) did not allow us to identify late sequelae, particularly disabling ones. It is essential to advocate for funding to conduct multi-center observational pilot studies to better analyse these lesions for improved management.\n\n\nConclusion\n\nThe incidence of blast-related severe craniofacial and ocular trauma at artisanal gold mining sites is increasing in Niger due to the proliferation of these sites. However, data on such injuries remain limited due to high pre-hospital mortality rates and their rarity. The absence of advanced neuroimaging techniques and limited healthcare resources further complicate the comprehensive care of these patients. The severity of the injuries and postoperative complications observed in our patient underscores the urgent need for therapeutic protocols tailored to resource-limited settings. Future clinical studies are essential to develop these protocols and improve patient management in such contexts.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/relative of the patient.\n\n\nAuthors’ contributions\n\nConceptualization: OIH, GI, AII, IDB, ABK, MSC, RS; Data Curation: OIH, IMD, FLA, KNA; Investigation: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Methodology: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Project Administration: GI, AII, IDB, ABK, MSC, RS; Resources: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Supervision: OIH, GI, AII, IDB, ABK, MSC, RS; Validation: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Visualization: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Writing – Original Draft Preparation: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS; Writing – Review & Editing: OIH, YBT, HAM, TMH, IMD, FLA, KNA, GI, AII, IDB, ABK, MSC, RS.", "appendix": "Data availability statement\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe authors are grateful to the following teacher-researchers: Pr. DECHAMBENOIT Gilbert, and the late Pr. SANOUSSI Samuila, for their unconditional support.\n\n\nReferences\n\nKallel Z, Maalej A, Fourati H, et al.: Évaluation médicolégale des blasts oculaires chez le militaire actif. J. Fr. Ophtalmol. 2022; 45: 928–936. PubMed Abstract \| Publisher Full Text\n\nKim SY, Yeh P-H, Ollinger JM, et al.: Military-related mild traumatic brain injury: clinical characteristics, advanced neuroimaging, and molecular mechanisms. Transl. Psychiatry. 2023; 13: 1–15.\n\nTovar MA, Bell RS, Neal CJ: Epidemiology of Blast Neurotrauma: A Meta-analysis of Blast Injury Patterns in the Military and Civilian Populations. World Neurosurg. 2021; 146: 308–314.e3. PubMed Abstract \| Publisher Full Text\n\nAlley MD, Schimizze BR, Son SF: Experimental modeling of explosive blast-related traumatic brain injuries. NeuroImage. 2011; 54: S45–S54. PubMed Abstract \| Publisher Full Text\n\nBryden DW, Tilghman JI, Hinds SR: Blast-Related Traumatic Brain Injury: Current Concepts and Research Considerations. J Exp Neurosci. 2019; 13: 117906951987221. Publisher Full Text\n\nZhang Y, Kang X, Wu Q, et al.: Explosive eye injuries: characteristics, traumatic mechanisms, and prognostic factors for poor visual outcomes. Mil. Med. Res. 2023; 10: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathews ZR, Koyfman A: Blast Injuries. J. Emerg. Med. 2015; 49: 573–587. Publisher Full Text\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. Publisher Full Text\n\nFeng K, Yao Y, Wang Z-J, et al.: Mechanism and prognostic indicators for explosion-related eye trauma: eye injury vitrectomy study. Acta Ophthalmol. 2021; 99: e956–e962. PubMed Abstract \| Publisher Full Text\n\nRosenfeld JV, McFarlane AC, Bragge P, et al.: Blast-related traumatic brain injury. Lancet Neurol. 2013; 12: 882–893. Publisher Full Text\n\nSachdeva T, Ganpule SG: Twenty Years of Blast-Induced Neurotrauma: Current State of Knowledge. Neurotrauma Rep. 2024; 5: 243–253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCourtney AC, Courtney MW: A thoracic mechanism of mild traumatic brain injury due to blast pressure waves. Med. Hypotheses. 2009; 72: 76–83. PubMed Abstract \| Publisher Full Text\n\nAlam M, Iqbal M, Khan A, et al.: Ocular injuries in blast victims. JPMA J. Pak. Med. Assoc. 2012; 62: 138–142.\n\nAbass Saley A, Baratoux D, Baratoux L, et al.: Evolution of the Koma Bangou Gold Panning Site (Niger) From 1984 to 2020 Using Landsat Imagery. Earth Space Sci. 2021; 8: e2021EA001879. Publisher Full Text\n\nCalys-Tagoe BNL, Ovadje L, Clarke E, et al.: Injury Profiles Associated with Artisanal and Small-Scale Gold Mining in Tarkwa, Ghana. Int. J. Environ. Res. Public Health. 2015; 12: 7922–7937. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAldag M, Armstrong RC, Bandak F, et al.: The Biological Basis of Chronic Traumatic Encephalopathy following Blast Injury: A Literature Review. J. Neurotrauma. 2017; 34: S-26–S-43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClausen AN, Bouchard HC, Mid-Atlantic VA, et al.: Assessment of Neuropsychological Function in Veterans With Blast-Related Mild Traumatic Brain Injury and Subconcussive Blast Exposure. Front. Psychol. 2021; 12: 686330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPermenter CM, Fernández-de Thomas RJ, Sherman AL: Postconcussive Syndrome. StatPearls. Treasure Island (FL): StatPearls Publishing; 2024 Jan. 2023 Aug 28. PubMed Abstract\n\nMcKee AC, Stein TD, Huber BR, et al.: Chronic traumatic encephalopathy (CTE): criteria for neuropathological diagnosis and relationship to repetitive head impacts. Acta Neuropathol. 2023; 145: 371–394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJennings N: Social and labour issues in small-scale mines: report for discussion at the tripartite meeting on social and labour issues in small-scale mines, Geneva, 1999. International Labour Organization; 1999.\n\nMounkaila A, Abdou DB, Yahaya M, et al.: Annuaire des statistiques sanitaires Niger, 2021. Direction des Statistiques du Ministère de la Santé Publique, de la Population et des Affaires Sociales, République du Niger.2021. Reference Source\n\nIssoufou Hamma O, Boka Tounga Y, Abdou Moussa H, et al.: Check list severe blast-related craniofacial and ocular injuries. Dataset. figshare. 2024. Publisher Full Text" }	[ { "id": "356101", "date": "14 Jan 2025", "name": "Mabel Banson", "expertise": [ "Reviewer Expertise Minimally invasive neurosurgery", "neurotrauma", "spine", "neurooncology", "hydrocephalus", "neuroendoscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes the management of a craniofacial with ocular injury following accidental explosion during clandestine gold mining activity. This report is of importance, not only in Niger, but also in other African countries where illegal mining is on the rise with its associated problems. The title is suitable and supported with a good abstract write-up. It is well discussed with backing literature. Please find below a couple of observations/comments for possible revisions.\nAbstract\nThe use of ‘reduced visual acuity’ may be qualified with specifying the eye involved with the mentioning of the fact that the left eye required enucleation. You may want to add ‘gold mining’ or similar to the list of key words\nIntroduction\nKindly reference the Second paragraph, line 3: “In our country … gold mining sites.” In the last paragraph, please elucidate what makes this case a rare one.\n\nIs it that few are listed in literature or the fact that the patient had only craniofacial injuries from a blast injury, or the patient had previous concussions from similar injuries, or the patient went back to mining following recovery, etc.?\nCase report\nParagraph 1, line 1-2: What was the patient’s premorbid state following the prior concussions from the explosions exposure? Paragraph 1, line 6: Was the distance to the health facility exactly 120km away? If not, you would want to describe it with the term ‘about’. What level of facility was the health center that the patient was sent to first? And, what level is the facility of the authors? It may also help understand the likely support available for such patients at the health center. Paragraph 2, line 1: Did you mean E1, V3, M5? Paragraph 2, line 8: Consider early mention of the type of material causing the penetrating injury. Paragraph 3:\nKindly explain what made the team utilize higher dosages for paracetamol and tramadol and not use other forms and more strong analgesics. Is it the common practice of the unit to use 96hour as prophylactic antibiotic treatment? The usual is up to 48hours, otherwise it stops being prophylaxis. The mention of “reference” and “reference range” can be made uniform by using just the term ‘reference’. What necessitated blood transfusion in this patient?\n\nPage 5, paragraph 1:\nConsider adding anesthetists/anesthesiologists to the list of multidisciplinary team as airway management is key in these set of patients. Were plastic surgeons involved in this case? You could mention the types of foreign bodies extracted. You would have to state here that there was a resulting cranial defect and give size if feasible. Why was a “limited surgical debridement” done in such a case? How long was the patient kept intubated? With reference to “On the third day, the patient was transferred to neurosurgery department”; is it the third day after injury/admission or after surgery?\n\nPage 5, paragraph 2\nWhat does the use of the term “clear consciousness” mean? Can you give Glasgow coma scale scores (GCS) and Glasgow outcome scores (GOS[e]) to elucidate the follow-up response better? The change of phenobarbital to sodium valproate: what was the reason? Was it a change of route of administration, or availability, etc.? You would want to qualify which third and fifth days that the examination of the surgical sites were done.\n\nPage 6\nDoes “Biological control tests” mean baseline tests for comparison? If so, it may not suffice here since there is suspicion of ongoing infection where it is being investigated; and initial lab results were already available. Consider revising to say, ‘septic screen’. Line 4: Describe further what is meant by “greasy dressing”. Were the follow up laboratory tests done? At the time of cessation of antibiotic therapy, what were the Hb, WBC, Platelets, ESR, CRP, etc. Was there any culture and sensitivity profile done for the likely organisms where the antibiotics used were changed to target the isolated organisms? Authors would need to indicate if it was a positive or negative culture or it was not done (with reasons). What kind of surgical revision was done? A debridement? Was there any local administration of antibiotics? Can you describe further what you mean by “fat dressings” The CT scan showing “good evolution” refers to what pathology on the preoperative images? What constituted the follow-up protocol? What outcomes were assessed in this patient during the follow-up period? What was the GCS and GOS(e) at discharge? What did “normal activity” mean in this patient? Do you have any post-operative pictures that show the healed injuries? It would be great to share if you have these.\n\nDiscussion\nCan you highlight some of the management challenges faced in this case? Since there is an advocacy angle to this write-up, the level of facility may be of importance to policy-makers. Kindly reference the first line of paragraph 1. Paragraph 1 line 8: At which point in time was the patient asked to recall the events of the injury? This patient had a compromised autonomy. Consider revising the statement. Eg: ‘The exact position of patient in relation to the dynamite explosion could not be ascertained’. Page 9, last paragraph:\nYou may want to mention a multidisciplinary approach under this area as part of the comprehensive management.\n\nLimitations: Clarify the first point. Is the number of cases referring to the case report given or the literature review.\nConclusions First and second lines would require referencing.\nFigures Figure 1\nIn A and B, is the foreign body obvious? If so, it would be good to mark it as well In the images, global rupture and disorganization of ocular structures is not obvious in the pictures. You may want to review the description. Also, the rhinorrhea is not as obvious; if you have a picture that shows it clearly, it may help, otherwise you may need to omit that description using this image.\nFigure 3\n3B: “lost-bone craniotomy” may be better described as ‘post-craniectomy defect’ 3D: The words “removal of” should be deleted with possible revision of sentence to ‘Granite fragments extracted from craniofacial injuries’\nFigure 4 The necrotic patch in picture B looks quite advanced. Is the use of the term early and advanced with reference to the time the necrosis developed? Kindly clarify. Figure 5 Is exposed part of the skull showing. If so, kindly mark it as well.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-855
https://f1000research.com/articles/13-273/v1	15 Apr 24	{ "type": "Data Note", "title": "Development and application of a learning enjoyment scale for pedagogical activities", "authors": [ "Tarig Merghani", "Rasha Babiker", "Azza O. Alawad", "Rasha Babiker", "Azza O. Alawad" ], "abstract": "The impact of learning enjoyment on motivation, enthusiasm, and overall learning experiences is significant. Previous studies, lacking an unbiased tool for measuring enjoyment and confronting various influencing factors, produced conflicting results regarding enjoyment levels in different instructional methods. Hence, we developed a learning enjoyment scale for evaluating both active and passive educational activities. We applied the developed scale to 112 first-year medical and dental students to assess their enjoyment during didactic physiology lectures and explored possible associated factors. Within this data note, we present students’ responses to the developed LES. The LES encompasses six dimensions: knowledge, comprehension, application, analysis, concentration, and enjoyment. Students provided ratings for each dimension on a five-point Likert scale, spanning from 1 (strongly disagree) to 5 (strongly agree). The cumulative scores across the six dimensions range from a minimum of 6 to a maximum of 30. These total scores can be categorized as excellent (> 24), acceptable (18-24), or low (< 18). The second section of the dataset examines specific factors influencing overall enjoyment, such as teacher proficiency, topic difficulty, active student participation, objectives fulfillment, low stress levels, and self-perceived acquisition of skills. In addition to objective measurement of students’ enjoyment level, the LES can be utilized for quantitative cross-comparisons between different teaching activities. By employing this dataset, we will undertake an analysis to determine the internal consistency of the Learning Enjoyment Scale (LES), with the anticipation that the outcomes will be published in another venue.", "keywords": [ "Pedagogical Activities", "Learning Enjoyment Scale", "Teaching Methods", "Didactic Lectures", "Teacher Proficiency" ], "content": "Introduction\n\nThe ongoing discourse on the effectiveness of teacher-centric learning approaches (passive instruction) versus student-centric learning methods (active instruction) persists, despite numerous studies conducted over the past three decades. Traditional lectures have been consistently criticized for their perceived lack of student engagement, largely due to their reliance on passive one-way information delivery. Students often view didactic lectures as dull or ineffective1–3 when compared to the active learning approaches. Consequently, many educators have embraced strategies such as incorporating real-life scenarios,4 introducing games,5 utilizing technology,6 or integrating problem-solving elements7 to enhance the enjoyability of their lectures. However, the challenge persists, as there are no universally clear guidelines on how to structure lectures that are both educational and enjoyable.\n\nConversely, students’ enjoyment may be influenced by various factors, including but not limited to the instructor’s skill, the nature of the topic, and the educational environment. Furthermore, the diverse personalities and learning styles of students all contribute to and impact the level of enjoyment experienced during a teaching activity.8–10\n\nDue to the intricate nature of these variables and the significance of enjoyment in the learning process, there is a pressing need for an objective measurement tool to gauge students’ enjoyment across various teaching activities. Such a tool would not only streamline the assessment of diverse teaching methods but also aid in pinpointing the factors contributing to fluctuations in enjoyment levels within the same teaching method when presented by different instructors or for different subjects by the same faculty. Consequently, we have devised a Learning Enjoyment Scale (LES) designed to evaluate both active and passive educational activities.1 In this study, we applied the developed scale to 112 first-year medical and dental students, evaluating their enjoyment during didactic Physiology lectures and investigating potential associated factors. This data note presents the students’ responses to the developed LES. Our newly created scale, coupled with the associated dataset,1 provides valuable insights into the nuances of student enjoyment, offering a tool that transcends the conventional dichotomy of teaching methods and delves into the factors shaping students’ learning experiences.\n\n\nMethods\n\nWe developed the Learning Enjoyment Scale (LES) as a comprehensive and objective tool for measuring students’ enjoyment in the learning process, Figure 1.1 The scale items are strategically aligned with the major categories of Bloom’s Taxonomy, emphasizing cognitive knowledge and affective attitude. Furthermore, the third domain, psychomotor skills, is partially assessed in the second section of the scale, highlighting its role as part of the overall enjoyment attributes. The LES first section consists of six items: knowledge, comprehension, application, analysis, concentration, and enjoyment. Students are instructed to rate each item on a five-point Likert scale, ranging from 1 to 5 (1 = strongly disagree, 2 = disagree, 3 = unsure, 4 = agree, and 5 = strongly agree). The minimum and total scores across the six items amount to 6 and 30, respectively. An excellent score is defined as above 80% (25-30), an acceptable score falls within the range of 60-80% (18-24), and a low score is considered less than 60% (<18).\n\nThe second section of the LES evaluates the impact of various factors on students’ enjoyment. These factors encompass the teacher’s talent or proficiency, the complexity of the topic, student participation, objectives fulfillment, perceived stress levels, and the development of skills. Through direct analysis, the contribution of each factor to the overall LES is determined, offering a detailed understanding of the specific elements influencing students’ enjoyment during the learning process. The scoring system of the LES questionnaire aims to provide a quantitative assessment of the multifaceted aspects contributing to students’ enjoyment in educational settings.\n\nThe provided dataset represents students’ responses to the items of the developed scale. The data was collected from first-year undergraduate medical and dental students during two physiology lectures. We selected first-year students as they are newcomers to university education, suggesting a lack of established preferences for specific instructional methods at this early stage. A total of 112 university students, age ≥ 18 y contributed, with an overall participation rate of 68%. Prior to completing the questionnaire, students provided written informed consent to participate. The University Research Ethics Committee (RAKMHSU-HEC) granted approval for the study on 15th November 2023, under the reference number “HEC-10-2023/24-F-M.”\n\nSeveral limitations need to be taken into consideration when interpreting the results of this study. Firstly, the exclusive inclusion of first-year students raises the possibility that their perceptions may differ from those of students in later academic years. Additionally, with a response rate of 68%, the potential impact of non-participating students’ perspectives on the results remains uncertain. Furthermore, the preference for didactic lectures may be influenced by the different learning styles of the students, which are not shown in this dataset.10 Lastly, additional investigation is warranted by applying this scale to different subjects, employing varied teaching methods, and exploring diverse geographic locations to enhance the generalizability of the scale.\n\nThe University Research Ethics Committee (RAKMHSU-HEC) granted approval for the study on 15th November 2023, under the reference number “HEC-10-2023/24-F-M.”\n\nStudents provided written informed consent to participate.", "appendix": "Data availability\n\nZenodo: Development and Application of a Learning Enjoyment Scale for Pedagogical Activities, https://doi.org/10.5281/zenodo.10526239. 11\n\nThis project contains the following underlying data:\n\n- Students’ responses to the developed LES: To assess students’ enjoyment during didactic lectures and explore possible associated factors.\n\nZenodo: Development and Application of a Learning Enjoyment Scale for Pedagogical Activities, https://doi.org/10.5281/zenodo.10526239. 11\n\nThis project contains the following extended data:\n\n- Learning Enjoyment Scale (LES): Developed for evaluating both active and passive educational activities.\n\nData are available under the terms of the Creative Commons Zero (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAlamoudi AA, Al Shawwa LA, Gad H, et al.: Team-based learning versus traditional didactic lectures in teaching clinical biochemistry at King Abdulaziz University; learning outcomes and student satisfaction. Biochem. Mol. Biol. Educ. 2021; 49: 546–559. PubMed Abstract \| Publisher Full Text\n\nSusanto T, Rasni H, Susumaningrum LA: The Comparing of Problem-Based Learning and Lecture-Based Learning on Students’ Learning Outcomes and Satisfaction for a Family Health Nursing Course. J. Keperawatan Padjadjaran. 2022; 10(2): 134–139. Publisher Full Text\n\nImran M, Halawa TF, Baig M, et al.: Team-based learning versus interactive lecture in achieving learning outcomes and improving clinical reasoning skills: a randomized crossover study. BMC Med. Educ. 2022; 22(1): 348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNarayanan SN, Merghani TH: Real-life scenario blended teaching approach for nurturing inquisitive learning of central nervous system in medical students. Adv. Physiol. Educ. 2023; 47(1): 124–138. PubMed Abstract \| Publisher Full Text\n\nNarayanan SN, Ahmed I, Saherawala B, et al.: Appraisal of a novel pedagogical approach to demonstrating neuromuscular transmission to medical students. Adv. Physiol. Educ. 2021 Sep 1; 45(3): 580–588. PubMed Abstract \| Publisher Full Text\n\nPremkumar K, Coupal C: Rules of engagement-12 tips for successful use of “clickers” in the classroom. Med. Teach. 2008; 30(2): 146–149. PubMed Abstract \| Publisher Full Text\n\nAlaagib NA, Musa OA, Saeed AM: Comparison of the effectiveness of lectures based on problems and traditional lectures in physiology teaching in Sudan. BMC Med. Educ. 2019 Sep 23; 19(1): 365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGifford H, Varatharaj A: The ELEPHANT criteria in medical education: can medical education be fun? Med. Teach. 2010; 32(3): 195–197. PubMed Abstract \| Publisher Full Text\n\nDeslauriers L, McCarty LS, Miller K, et al.: Measuring actual learning versus feeling of learning in response to being actively engaged in the classroom. Proc. Natl. Acad. Sci. USA. 2019; 116(39): 19251–19257. Publisher Full Text\n\nBuşan AM: Learning styles of medical students - implications in education. Curr. Health Sci. J. 2014; 40(2): 104–110. PubMed Abstract \| Publisher Full Text\n\nMerghani T, Babiker R, Alawad A: Development and Application of a Learning Enjoyment Scale for Pedagogical Activities. [dataset]. Zenodo. 2024. Publisher Full Text" }	[ { "id": "268529", "date": "17 May 2024", "name": "Tarig Fadelelmoula", "expertise": [ "Reviewer Expertise Pulmonology", "Physiology", "Medical Education", "Healthcare Simulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExisting student learning enjoyment evaluation scales are not sufficiently tested. The objective of this project is to develop and apply a new learning enjoyment scale for evaluating students’ enjoyment during different pedagogy sessions. The researchers designed the tool in accordance with Bloom's taxonomy. The tool had two sections: (1) A six-domain Likert scale, (2) examining specific factors influencing overall enjoyment, such as teacher proficiency, topic difficulty, active student participation, objectives fulfillment, low stress levels, and self-perceived acquisition of skills. The scale was implemented in year 1 medical and dental students. The initial analysis of data reflected the internal consistency of the scale; however, the results are limited by the low response rate, the confinement to didactic lectures, and student level. Further research is needed to include a larger group of students, check the reliability of the scale, and test it in student centered pedagogies.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11750", "date": "11 Jun 2024", "name": "Tarig Merghani", "role": "Author Response", "response": "Many thanks for the review comments. We appreciate your insights and feedback on our data note. Further results and analysis will be presented in a full article (under review). We acknowledge that existing student learning enjoyment evaluation scales have not been sufficiently tested, which is why our project aimed to fill this gap by designing a comprehensive tool based on Bloom's taxonomy. We are pleased that the initial analysis reflected the internal consistency of the scale. However, we also recognize the limitations you pointed out, namely the low response rate, the confinement to didactic lectures, and the restriction to year 1 medical and dental students. We are planning to address these limitations by including a larger and more diverse group of students. Thank you once again for your valuable feedback. Regards Tarig Merghani" } ] }, { "id": "300476", "date": "22 Jul 2024", "name": "Mohi Eldin Magzoub", "expertise": [ "Reviewer Expertise This article aims at developing a useful tool for measuring student engagement and satisfaction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 introduced a new instrument called the Learning Enjoyment Scale, which is used to assess the degree of enjoyment experienced by students. It is derived from Bloom's taxonomy. The scale encompasses the majority of the taxonomy categories on the knowledge domain and few items on the affective domain. Nevertheless, the scale primarily serves to categorize learning objectives in order to facilitate course design and direct assessment. Thus, it aims to enhance the overall efficacy of the learning and teaching process, and not directly put an emphasis on enjoyment. Although enjoyment is not explicitly stated as an objective in the affective domain, it can be seen as a constituent of favorable attitudes and values towards learning. The affective domain encompasses various categories, such as perceiving events, reacting to phenomena, assigning value, organizing, and characterizing based on a set of values. Enjoyment can be understood as the process of appreciating and reacting to various experiences, where the active involvement and curiosity of students in educational tasks play a significant role. There are only four items in the scale that directly focus on enjoyment and satisfaction, which are questions 5, 6, 11, and 12. Other learning theories might be explored that nest suit student enjoyment such as experiential l learning self-determination, and constructivism theories. Another issue with the methodology is the lack of clear objectives. It is unclear whether the goal is to validate the scale by assessing reliability and validity, or to present the performance of students based on the items of the scale. The objectives must be explicitly stated and appropriately included into this section. Another concern is how the cut-off points for determining excellence, poor, and low are established. The utilization of the five-point Likert scale is not suitable in this context. The scale must adhere to a linear format instead than a categorized format. For example, a value of 3 for \"unsure\" indicates that it cannot be decided whether it is high or low. An optimal approach would be to employ a rating scale consisting of either 5 or 10 points, including a spectrum from poor to excellent. Subsequently, a composite score can be calculated based on this scale.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "12077", "date": "31 Jul 2024", "name": "Tarig Merghani", "role": "Author Response", "response": "Thank you very much for the comprehensive review and valuable comments. We have carefully considered the points you raised and prepared an updated version of our data note (version 2). Please, find our detailed responses to the raised points below. Student Enjoyment is a state of psychological happiness experienced during learning. It is nicely defined by Hartley as the pleasant emotional state of the learner that develops during an educational activity due to experiencing a positive situation that motivates the learner to complete the task to persevere this feeling (1). It is different from engagement, satisfaction or motivation. Since it is subjective feeling, there is no widely accepted scale for assessing enjoyment. Authors tried different methods ranging from a single question rating (for example from 0 to 6) (2), to different types of questionnaires such as satisfaction questionnaire with open ended questions (3, 4), adapted items from Achievement Emotions Questionnaire (5) and researcher-prepared questionnaire based on enjoyment indicators (6). In our scale, we utilized direct questions to measure enjoyment, and we added other questions to assess the students’ perception of different domains of learning (knowledge, comprehension, application, and analysis) which are known to be influenced by enjoyment (Q1 to 6). Other questions (Q7 to 12) are not intended to measure the enjoyment, they are intended to study certain factors that are known to influence enjoyment. The primary objective of this study is to develop a comprehensive learning enjoyment scale for the evaluation of both active and passive teaching activities. We sought to apply this scale objectively to measure the enjoyment scores of Physiology lectures and investigate associated factors. As suggested by the reviewer, we clearly mentioned the objectives of our study in the updated version of the article. The suggestion to explore other learning theories like experiential learning, self-determination theory, and constructivism is well-taken. We plan to incorporate discussions on how these theories could complement our findings on student enjoyment and educational outcomes in future studies. We also have the idea to revise and update our scale in the future based on feedback from reviewers and valuable suggestions from colleagues. The Learning Enjoyment Scale (LES) employs a five-point Likert scale (1 = strongly disagree to 5 = strongly agree) across its six dimensions, yielding total scores ranging from 6 to 30. The 80% represents the fourth option of the Likert scale (agree = 4), as 4/5 x 100%= 80%. Therefore, scores above 80% generally indicate that all responses are agree or above. The 60% represents the third option (unsure= 3), as 3/5 x 100%= 60%. Therefore, scores below 60% generally indicate that all the responses are disagree or below. The 5-Likert scale selection and categorization into excellent, acceptable, and low is justified by its ability to provide clear interpretations of students' enjoyment levels. In addition, the thresholds (60% and 80%) align with educational standards commonly used to assess performance and satisfaction levels in various academic contexts. In conclusion, we believe that our scale contributes significantly to the medical education field by addressing the gap in measuring student enjoyment in educational contexts. We appreciate the opportunity to revise our manuscript based on the reviewer's valuable feedback and look forward to sharing our full manuscript that contains enhanced findings, analysis and discussion with the academic community in the near future. Thank you once again for your thorough evaluation and insightful comments. Best regards, Tarig Merghani References: Hartley D. Excellence and enjoyment: The logic of a ‘contradiction’. British Journal of Educational Studies 2006; 54(1):3-14. https://doi.org/10.1111/j.1467-8527.2005.00331.x Griffee DT. Connecting Theory to Practice: Evaluating a Brain-based Writing Curriculum. Learning Assistance Review 2007;12(1):17-27 Marín-Vinuesa LM, Rojas-García P. Expected Usefulness of Interactive Learning Platforms and Academic Sustainability Performance: The Moderator Role of Student Enjoyment. Sustainability. 2024; 16(9):3630. https://doi.org/10.3390/su16093630 Dehghan S, Horan EM, Frome G. Investigating the Impact of the Flipped Classroom on Student Learning and Enjoyment in an Organic Chemistry Course. Journal of Chemical Education 2022; 99(7):2512-2519. https://doi.org/10.1021/acs.jchemed.1c01104 Bieleke M, Gogol K, Goetz T, Daniels L, Pekrun R. The AEQ-S: a short version of the achievement emotions questionnaire. Contemp. Educ. Psychol. 2021; 65:101940. https://doi.org/10.1016/j.cedpsych.2020.101940 Mirawati M, Sikarni W. Description of Student Attitudes: Enjoyment in Learning Physics and Interest in More Time Studying Physics. Sch. Jo. Phs. Ed [Internet]. 2023 Mar.31 [cited 2024Jul.24];4(1):1-. Available from: https://cahaya-ic.com/index.php/SJPE/article/view/490" } ] } ]	1	https://f1000research.com/articles/13-273
https://f1000research.com/articles/13-851/v1	30 Jul 24	{ "type": "Research Article", "title": "Trust in open publishing practices", "authors": [ "Eva Kalmar", "Thijs Elzer", "Nicoleta Nastase", "Thirza Bolhuis", "Nathaniel Germain", "Maurits Rietveld", "Yiwei Tao", "Sophie de Wolff", "Thijs Elzer", "Nicoleta Nastase", "Thirza Bolhuis", "Nathaniel Germain", "Maurits Rietveld", "Yiwei Tao", "Sophie de Wolff" ], "abstract": "Background Scientific publishing is a critical part of scientific enquiry; individual excellence is often measured by the number of publications, and the journals in which these publications appeared count enormously. Open Science practices, such as open access, open review, random gatekeeping and shared governance, are implemented in various journals and publishing platforms, providing alternative ways of publishing. But how are these practices trusted?\n\nMethods We have created a theoretical framework for trust in the context of academic publishing and investigated to what extent Dutch researchers find these ways of publishing trustworthy. We have performed a survey to compare the trustworthiness of novel and classical ways of publishing and conducted multiple interviews to figure out why scientists find certain publishing practices more attractive than others.\n\nResults In the academic publishing system, we have identified various interdependent relationships between stakeholders in which there is a certain level of uncertainty; therefore, we can assume that trust plays a relevant role here. Based on the literature review and interview results, trustworthiness turned out to be one of the most important factors in choosing journals to read relevant scientific discoveries and to publish papers. The survey results suggest that some aspects of open publishing, such as open access, open peer review and shared governance are well-known and widely accepted and trusted amongst the participants, while others, like participatory peer review or random gatekeeping, were less known. In these cases, many researchers formulated concerns about the competence of the randomly assigned gatekeeper and the reviewers coming from the wider community.\n\nConclusions Our results highlight a shift in social norms within the Dutch scientific community, formulating critical voices towards the profit-oriented nature of classical scientific publishing and highlighting the importance of open access to scientific results, supporting open peer review and publishers with shared governance.", "keywords": [ "Open Science", "scientific publishing", "trust", "open access", "peer review", "governance", "gatekeeping" ], "content": "Introduction\n\nIn 2021, UNESCO created a Recommendation on Open Science, which is defined as “a set of principles and practices that aim to make scientific research from all fields accessible to everyone for the benefit of scientists and society as a whole (UNESCO).”\n\nThe Open Science movement focuses on, among others, the accessibility of scientific results in the form of publications, data, source materials and digital representations (Sönke Bartling & Friesike, 2014). Free access to the generated scientific knowledge is claimed to be the key to innovation and human development (Cribb & Sari, 2010; Phelps, Fox, & Marincola, 2012), a democratic right of every taxpayer by making publicly funded research publicly accessible (Phelps et al., 2012), and a means to increase the replicability and reliability of scientific records and reduce publication bias (Reynolds, 2016). Sharing scientific results can happen in various ways, from scientific publications through conference presentations, seminar discussions, digital conversations, mail exchanges, writing blogs, and posting on social media. Some scholars even suggested sharing timestamped and referrable ideas and negative or preliminary scientific data (Bartling & Fecher, 2016). Still, researchers are often afraid to share their unpublished results or ideas due to their fear of scooping, not being credited for ideas, public humiliation, risk to reputation, reduced scientific quality, career compromise, backlash from senior figures, or simply being different (Gomes et al., 2022). Therefore, scientific results are still disseminated mainly through publishing them in scientific journals and books (Schimanski & Alperin, 2018).\n\nPublishing practices and policies enormously shape and determine the other phases of the scientific life cycle through the reward structure coupled with publications. Although there are initiatives to get rid of the impact factor and H-index (like recommended by the San Francisco Declaration on Research Assessment - DORA), in most countries, in one way or another, the scientific publication list is still one of the most important factors to determine the academic impact of a scientist, leaving out leadership, vision, teaching qualities, teamwork or collaboration out of measuring excellence, for example in evaluating grant applications (Borycz et al., 2023; Khan, Almoli, Franco, & Moher, 2022; McKiernan et al., 2019; Morton, Ranson, & El-Boghdadly, 2023). The consequences of this are the well-described publish or perish phenomenon (Lee, 2014), the unequal distribution of resources to institutions and researchers that are already successful (Bezuidenhout & Chakauya, 2018; Onie, 2020), and avoiding investing in risky but innovative projects or financing novice researchers with original ideas (Stephan, 1996).\n\nOne of the most claimed issues of the current publishing system is the subscription-based business model, in which the authors and the peer reviewers do not receive any monetary incentives for their intellectual work, but the readers have to subscribe to the journal or pay for the article individually (Tennant et al., 2017). Making the final version of scientific publications permanently and freely accessible to everyone (open access) can be achieved by journals in several ways. One often-used version of open access includes article processing charges (APCs), which are paid for by authors prior to publication, often from research grants (Owens, 2003). This type of open access, called the gold open access, has become one of the most widespread manifestations of open science. At the end of 2021, the global open access level for research published reached 49%, most of which are gold open access publications (Neylon & Huang, 2022). The introduction of APCs reinforced a different type of publication bias: authors from less-resourced fields, countries, or organisations publish less this way (Borrego, 2023; Klebel & Ross-Hellauer, 2023; Nabyonga-Orem, Asamani, Nyirenda, & Abimbola, 2020). Various organisations support full open access to research publications funded by public or private grants (“cOAlition S”) by using non-profit ways of academic publishing, called the diamond open access (Fuchs & Sandoval, 2013).\n\nPublishing preprints, self-archiving (also called green open access), and making datasets available and reusable in repositories (Ware & Mabe, 2015) are aimed at solving some other issues related to the current mainstream way of scholarly publishing, such as not acknowledging peer reviewers, the lack of reproducibility (van Rossum, 2017), the low incentives to publish negative results, and the lack of reusability of published work (Niya et al., 2019). Several other approaches aim to change these deeper aspects of academic publishing, providing alternative ways of peer review, such as publishing the peer review together with the article (public review or open peer review) or inviting a broader group from the scientific community to provide feedback to the authors (participatory review or open participation peer review) (Ross-Hellauer, 2017; Walker & Rocha da Silva, 2015), using random gatekeepers instead of editors (Tennant et al., 2017), or trying out shared governance over private companies or decentralised infrastructure (Tenorio-Fornés, Tirador, Sánchez-Ruiz, & Hassan, 2021).\n\nDifferent journals and publishing platforms have various combinations of these solutions, applying incremental changes in their practices or introducing revolutionary new ways of academic publishing. Of course, the number of publications appearing on these platforms and in these journals provides us with some information about how many scientists are eager to change their publishing practices, but we do not know for sure what scientists still publishing in “traditional” journals think about these “new” practices.\n\nAt the same time, although there are critical points of the current mainstream way of publishing, it is still highly accepted and trusted by the broad scientific community (Tenopir, 2014). Due to the spread of predatory journals, which exploit the current academic system based on the publish or perish principle and deceive potential authors to make them pay for publishing their results in journals that do not provide traditional peer review and editing (Shrestha, 2021), trust in the classical way of publishing might even be strengthened.\n\nWe were therefore interested in to what extent scientists trust journals and publishing platforms that implement aspects of open science. We first created a theoretical framework to define and conceptualise trust for this context, then investigated the academic publishing system from the perspective of this framework to answer the sub-question: What kind of role does trust play in academic publishing? Then, we asked researchers at one TU Delft faculty to generate ideas and to come up with possible solutions to these issues of academic publishing. We have used the theoretical framework to dissect trust into smaller and more tangible concepts, such as benevolence, competence, integrity and predictability, and we investigated to what extent researchers and PhD candidates working in Dutch research institutes trust the aspects of the new publishing practices such as open access, open review, random gatekeeping and shared governance.\n\n\nMethods\n\nA mixed-methods strategy was chosen to answer the research question: To what extent do researchers trust new initiatives in academic publishing? First, we performed desk research to create a framework for trust and investigated the various trust relationships in academic publishing. Then, we invited several TU Delft-based researchers to generate potential solutions for some issues related to academic publishing. Based on the themes that emerged from this session and the theoretical framework, we created a quantitative online survey to get a general picture of to what extent researchers would trust various aspects of academic publishing. This was followed by qualitative semi-structured interviews with researchers from various Dutch universities to gain a deeper understanding of the reasons why they trust or distrust several aspects of open science.\n\nThe literature study, the ideation session, and the B interview series were conducted by a researcher with an engineering and social scientific educational background together with a researcher with a design research background. The survey and interview series A were performed by a team of Master students following the Research Methods course of the Communication Design for Innovation Master track at TU Delft, under the supervision of a researcher with social scientific research background.\n\nBecause our research question focused on researchers involved in the academic publishing system, both on the user and production sides, we decided to include researchers who have publishing, peer reviewing and editing experience. To focus the research to out local environment, we decided to invite Dutch researchers.\n\nIn total, sixty researchers filled out the survey completely. They represented social sciences, humanities, natural sciences, and engineering disciplines. More than half of the people who filled in the survey were at the beginning of their career; 20% had ten to nineteen years of work experience, while 25% had worked in science for over 20 years. Most participants (95%) have already published scientific publications. Two-thirds of the participants had less than 24 articles published, while almost 14% had more than 75 publications. 78% of the participants have already performed a peer review before, and 20% were involved in the peer review of more than 75 papers.\n\nTen researchers from one TUD faculty were asked to participate in the ideation workshop. The group consisted of PhD students, assistant, associate, and full professors with various expertise in publishing and peer reviewing. Four participants from various Dutch universities were interviewed in Interview Series A, and eight researchers from TU Delft in Interview Series B. They worked at various faculties and held positions ranging from assistant, associate and full professor.\n\nAll parts of the study, including the informed consent forms, the survey, the interviews and the idea generation session, were approved by the Human Research Ethics Committee (HREC) of TU Delft prior to the given part of the research (Letter of approval #2196, signed on the 12th of May 2022 by the Chair HREC TU Delft and Letter of approval #3180, signed on the 19th of May 2023 by the Chair HREC, TU Delft). Participants were fully informed about the research, including the purpose of the study and how their responses would be used. Any potential risks or benefits of participating were also explained clearly, and it was emphasised that participation was voluntary and that participants could withdraw from the study until the data collection was finished. A formal consent form was provided, previously approved by the ethical committee, which all participants were required to verbally agree to (in case of online interviews, in which case the verbal consent was recorded and stored as an audio file) or sign before the idea generation session and interviews. Both types of informed consent (written and verbal) for the surveys and interviews are accepted standard methods to obtain consent and were approved by the ethical committee. As for the online interviews the participants were not present, we have chosen to ask their verbal consent so that the participants did not have to send the signed consent forms via email, including one more risk for data leakage. In the case of verbal consent, the audio recordings were kept in a separate secure folder. The survey started with a short information about the research, explaining the aim of the research, the duration of filling in the survey, the confidential nature of the data collection, the data management process, the potential risks of participating in the research, the voluntary participation and the contact information of the responsible researcher. After this introduction all participants had to agree with these conditions, otherwise their data were not collected. Please find the exact introduction page in the survey document (Kalmar, 2024b). All collected data was stored securely in Microsoft Teams with two-factor authentication, and only group members and the responsible researcher had access. Besides, all personally identifiable data, such as name, IP address and response date, were removed from the dataset during the data cleaning and analysis stages.\n\nDesk research\n\nFor the theoretical framework, we performed the first round of literature search with trust-related terms in Google Scholar because of its broad coverage of reports and books (Martín-Martín, Orduna-Malea, Thelwall, & Delgado-López-Cózar, 2019). After the initial search, we used the results to refine the search queries and snowball additional sources. Search terms included trust and trustworthiness, distrust, mistrust and untrust, trustworthiness, vulnerability, risk, threat, uncertainty, dependence, and control. From the included sources, clusters of similar information were formed, and these clusters were enriched with additional information from subsequent literature searches (e.g., by doing an in-depth search into the meaning of uncertainty, which is one of the trust conditions).\n\nThe second round of literature review was performed to link the theory in the framework with actual examples from the academic publishing world. For this, we used the Web of Science. The following search terms were used in various combinations: goals, dependence and uncertainty; academic, scholar or scientific publishing; and finally, readers (audience, public), authors (scientists, researchers), editors, peer reviewers (referees), librarians (libraries, universities) and publishers. We have selected articles that turned out to be relevant upon reading the abstract, read the article and summed them up.\n\nIdeation session\n\nTen TU Delft researchers were invited to brainstorm on possible solutions for problems in academic publishing related to the duration of the manuscript processing, the professionalism in peer review and the power of researchers relative to major publishers. During the first phase, the workshop organisers presented the problems and shared some insights related to the issues. The researchers were asked to generate as many ideas as possible. By rotation, all researchers came across all three problem areas, and each of them went through two iterations of idea generation. During the second phase, the researchers were invited to collect all the ideas, discuss them, put them into various clusters, and then rank the categories based on their importance.\n\nSurvey\n\nWe created a cross-sectional quantitative online survey using Qualtrics. It contained 47 questions and took approximately 20 to 30 minutes to complete. Before distribution, a pilot survey was conducted with colleagues, and based on their feedback and comments, the survey was adjusted.\n\nThe participants were asked to rate their trust in various aspects of academic publishing, such as open access, gatekeeping, peer review, and governance. They had to share to what extent they agreed with statements related to general trust and the trust concepts of competence, predictability, benevolence and integrity (see Table 1 for their definitions). In the case of access, only integrity and benevolence were used. The questions in this part of the survey were answered using a Likert scale with five agreement options ranging from Totally disagree to Totally agree, also inducing a Not Applicable (NA) option.\n\nParticipants were recruited via email and newsletters from university-specific graduate schools and open science institutions in the Netherlands. The survey was conducted online from May 9th to June 5th 2023, and 91 responses were received, of which 60 were complete and subject to further analysis.\n\nThe questionnaire text, including the questions is made available for further use at the DANS data repository (Kalmar, 2024b).\n\nInterviews\n\nInterview series A: Four semi-structured interviews were conducted with survey respondents who indicated interest in participating. The interviews were conducted to understand better why some participants trust specific options for the publishing attributes. These interviews started with an introduction to the topic, the research, and the research goal. Each interview took about 30 minutes and consisted of several questions about the interviewee’s opinions on academic publishing, as well as their opinions regarding trust. There were no questions related to the specific answers they gave in the survey, but they were encouraged to give reasons why they trust certain aspects more. Interviews were conducted online using Microsoft Teams. Draft transcripts were provided directly from the app and manually cleaned and coded later. Video recordings were securely stored on the university-provided server and were deleted after transcription. The full protocol for the interview series A is made available in the DANS repository.\n\nInterview series B: Furthermore, we performed semi-structured interviews with eight researchers from TU Delft (who did not participate in the survey research) to ask about their publishing preferences. These interviews lasted approximately an hour and were performed using Microsoft Teams. The full protocol for the interview series B is made available in the DANS repository.\n\nIdeation session\n\nThe generated papers were collected and photographed, then the cateogires were manually analysed.\n\nSurvey\n\nThe questionnaire data was first manually cleaned, unfinished answered were excluded from the analysis. Then all personal information (such as IP address and email address) were deleted from the answers. Then the remaining data was subject to descriptive analysis by using SPSS. The Totally Agree and Agree Likert-scale answers were merged into the Positive Answer category. At the same time, Totally Disagree, and Disagree options were merged into the Negative answer category for the analysis. Then, using the Wilcoxon signed-rank test, we performed statistical analysis on the dataset to determine which options for each publishing attribute the participants trusted more.\n\nThe anonymised dataset collected via the survey is made available for further use at the DANS data repository (Kalmar, 2024a).\n\nInterviews\n\nAll interviews were transcribed and subject to thematic analysis. Provisional and open coding were used in the first round of the analysis by using Atlas Ti, and codes were grouped into code families in the second round (Wertz, 2014).\n\n\nResults\n\nThis article uses the following definition of trust: “trust is an attitude that one takes to the trustworthiness of another; in turn, the other’s trustworthiness is a property that they have” (O’Hara, 2012). Trust, from this perspective, is a relationship between trustors (the trusting persons or entities) and the trustees (the trusted persons or entities) (Nooteboom & Six, 2003). Furthermore, trust is needed when the trustors are dependent upon the trustees, the trustees’ resources or their access to resources for the realisation of the trustors’ goals (Davis & Cobb, 2010) and there is uncertainty about whether the trustees will or can fulfil the trustors’ expectations (Gambetta, 2000).\n\nThe trustors decide to trust the trustees directly or via a mediator, also called a trust broker (Nooteboom & Six, 2003). Direct trustees can be actors but also inanimate or intangible objects like data, information, knowledge (Hardwig, 1991), processes (Eshuis & Van Woerkum, 2003), code, quasi-entities and algorithmic authorities (Teng, 2021). The act of trusting can be a conscious decision or based on non-rational clues, such as previous experiences, emotions or moods. The trustors act on their beliefs and/or decisions by following through with trust-informed risk-taking behaviours (Dietz & Den Hartog, 2006). Trustors often consider the trustee’s benevolence, competence or ability, integrity and predictability when determining the trustee’s trustworthiness. Table 1 contains the working definitions of these various trust elements.\n\nIn any academic publishing system, trustors and trustees may include (but are not limited to) readers, authors, peer reviewers, editors, publishers and librarians. Researchers can take on almost all trustor and trustee roles. Trustees may also come in the form of proxy trust metrics (such as impact factors) and research outputs, such as peer-reviewed scientific articles, conference proceedings, preprints, monographs, edited volumes, books, non-peer-reviewed grey literature, magazines and trade journals, reference works, blogs and social media posts (Nicholas et al., 2015). In academic publishing, the role of a third-party trustee is, amongst others, fulfilled by publishing groups, including publishers and journals. Publishers try to establish trust through their services by date-stamping or priority via registration, quality-stamping (certification) through peer review, recording the final, definitive and authorised versions of papers, archiving them and disseminating these to targeted scholarly audiences (Hummels & Roosendaal, 2001).\n\nBecause the academic impact of researchers is still often measured in citation indexes (Ravenscroft, Liakata, Clare, & Duma, 2017; Tenopir, 2014) and other altmetrics, such as the number of tweets and blogs mentioning the published paper and the number of times it is bookmarked (Williams, 2017), authors of scientific publications depend upon readers downloading and citing their articles. When choosing a journal to publish, authors often have their audience in mind (Tenopir, 2014). In the last couple of decades, it became more and more important to publish papers that reach “enough” attention (Van Noorden, 2017). Therefore, researchers in some fields depend on prestigious publishers (through prestigious journals) (Larivière, Haustein, & Mongeon, 2015), however, the trustworthiness of these journals is also questioned (Brembs, 2018). These prestigious journals are praised, therefore, it is highly uncertain whether authors can land their papers in such journals; their rejection rates range from 80% to 95% (Khadilkar, 2018). Due to this phenomenon, most researchers aim not for these journals but for those that promise more chances to be published. For some researchers, the citation count and the likelihood of getting published are more important than the trustworthiness of the journal because of the pressure of moving forward in their academic career (Tenopir, 2014).\n\nOpen access can increase the audience reach of a paper (Taylor & Francis, 2019) and the number of citations (Huang et al., 2024; McKiernan et al., 2016), therefore, could be a preferred way of publishing. Nevertheless, some early studies investigating the trust in citing behaviour of researchers found that open access journals were less trustworthy compared to closed access journals (Watkinson et al., 2016). The reasons behind distrusting open access journals are multifold, but doubting the quality of open access journals due to the spread of predatory journals is one of the major causes (Editage, 2018; Watkinson et al., 2016).\n\nReaders are dependent on authors to gather, process and present information in a scientifically sound way. “Whenever the relevant evidence becomes too extensive or too complex for any one person to gather it all […] one can have sufficient evidence only through testimony” (Hardwig, 1991). According to the Enago Academy’s report, yearly 500-600 academic papers are retracted because of inconsistencies caused by honest errors, biases or deliberate scientific misconduct (EnagoAcademy, 2021). Readers often trust articles recommended by colleagues (posted on social media or blogs). Familiar journals are also considered trustworthy, and in the case of unfamiliar journals, readers award more credit to peer-reviewed journals and journals with higher impact factors. Some readers check the trustworthiness of papers published in unknown journals by critically evaluating the abstract, the methods, the data published in the article and the references (Tenopir, 2014).\n\nDue to their role in quality control, editors are often called the gatekeepers of academic publishing, although calling them custodians may be more appropriate (Starfield & Paltridge, 2019). Readers depend on editors’ judgment to reject low-quality papers and ensure that the accepted papers comply with specific standards. In addition, readers depend on editors to correct already published work, release an expression of concern about it, or retract it (Vaught, Jordan, & Bastian, 2017) when the paper contains questionable results.\n\nEditorial boards have a huge responsibility to judge the quality and integrity of the scientific research reported in the manuscripts. Unfortunately, editorial boards were found to have extremely low levels of diversity. Only 20% of editorial board members were women, 1% were underrepresented minorities, and more than 25% of the editors came from less than 5% of the research institutes (Liu, Rahwan, & AlShebli, 2023; Newhouse & Brandeau, 2021).\n\nThe gatekeeping activity of editors is often outsourced to or shared with peer reviewers, creating a dependent relationship between editors and reviewers (Jackson et al., 2021). Finding peer reviewers to provide feedback on particular papers is often challenging for editors. This makes them more dependent on authors. Although controversial, journals frequently ask authors to suggest peer reviewers when they submit their manuscripts (EnagoAcademy, 2021). However, this also opens the door to peer review circles and citation rings, in which peer reviewers provide favourable comments to each other, and authors write their own peer reviews (Ferguson, Marcus, & Oransky, 2014). Such fake peer reviewers accounted for 15% of the retracted papers (Kulkarni, 2016).\n\nAuthors are dependent on the editors to publish their papers. At the same time, they cannot be confident about the fairness of the decision-making process because editors bring subjectivity to the process by making the final decision and choosing peer reviewers (Primack et al., 2019). In addition, authors may be uncertain about what exactly is happening to their manuscripts because journals/editors do not always communicate transparently during the manuscript-handling process (Taylor & Francis, 2015). Authors, in general, want high-quality feedback on their manuscripts, feedback that is specific, actionable, digestible, reasonable, respectful and consistent (Gerwing et al., 2020; Lanier, 2021; Omer & Abdularhim, 2017; Resnik & Elmore, 2016). They also want the contents of their manuscripts to be handled with discretion because “being scooped to a discovery is a scientist’s worst nightmare” (Callaway, 2019). Finally, authors may be uncertain whether peer reviewers act in their interests. In several instances, reviewers have been shown to be biased against papers from certain groups of people (female, non-native English authors or junior researchers, authors from specific geographical locations or low-prestige institutions) (Amano et al., 2023; Fox, Meyer, & Aimé, 2023; Liu et al., 2023; Nakamura et al., 2021; Taylor & Francis, 2015; Walker & Rocha da Silva, 2015). Even knowing the flaws of the system, peer review was found to be trusted by readers and authors to provide authority, quality and reliability (Watkinson et al., 2016).\n\nIn 2020, there were roughly 600 publishing platforms and journals which have implemented various versions of the open peer review system (Wolfram, Wang, Hembree, & Park, 2020). Open peer review reveals the content of the peer review, the identity of the referees before or after the publication of the manuscript and/or opens up who can review or comment on the publication (Ross-Hellauer, Deppe, & Schmidt, 2017). This system is aimed to provide more transparency, enrich scientific records, while honouring peer reviewers, and increase the review quality and integrity by reducing reviewer bias and decreasing the manuscript handover time (Tennant et al., 2017). Open peer review requires and builds on mutual trust between authors and reviewers (Schmidt, Ross-Hellauer, van Edig, & Moylan, 2018). Therefore, it is crucial for the wide application of open peer review practices that researchers trust these processes.\n\nFigure 1 depicts the theoretical framework of trust, and the various stakeholders found relevant related to trust in academic publishing.\n\nDuring the idea generation session, ten researchers from TU Delft were asked to generate solutions for three areas of academic publishing: the duration of manuscript processing, professionalism in peer review, and the power of researchers relative to major publishers. They have clustered the solutions into categories, and we identified the following aspects of the publishing process as most important for TU Delft researchers: open access practices, new governance models, different proposals for editorial control of the peer-review process and for the reviewers’ support.\n\nOpen access\n\nSeveral researchers suggested reforming the system and making all publications open access as a solution. Until that happens, they suggested that pirate sites, which provide open access to articles otherwise behind a paywall in an illegal way, should be accepted. Some participants suggested creating a campaign to collectively publish in open access minor journals as a protest against profit-oriented publishers or even banning publishers still applying this publishing model. Several people suggested promoting publishing preprints.\n\nPeer review\n\nResearchers proposed multiple directions to improve the peer review system, including public review (publishing the review together with the publication or publishing the review in a separate journal edition), providing an online one-on-one session for authors and reviewers after the written review to discuss the review in detail, publishing the rebuttals for reviews also publicly, or having a rating or evaluation system for peer reviewers based on the quality of their feedback. Some researchers brought up the idea of integrating an automatic process in the peer review, by implementing an AI-based pre-review, for example.\n\nGatekeeping\n\nResearchers thought that publishers or platforms need to invest in hiring more people to do peer reviews, provide (sensitivity) training for peer reviewers, change the editorial boards more frequently and set up and clearly communicate concrete deadlines for the manuscript processing. They also came up with ideas to filter out inappropriate reviews.\n\nGovernance\n\nThe participants of the idea generation session suggested the wider introduction of the shared ownership structure in publishers and publishing platforms, creating more university-based journals, and having more state-owned or crowdfunded publishing platforms.\n\nReward and recognition\n\nBesides improving the publishing process, we received suggestions of intervention through recognition and reward mechanisms, like reputation scores—both positive and negative—and punishment mechanisms for the lack of research integrity. Lastly, we received suggestions on diversifying the paper size and format to ease the publishing process.\n\nOur literature study showed that trust relationships and interdependencies exist between the various stakeholders of academic publishing. Based on the categories that emerged during the idea selection stage of the ideation session, we have decided to focus on the following aspects of open publishing during the rest of the research: open access, peer review, gatekeeping, and governance. To investigate how researchers at various Dutch universities trust these aspects of open publishing, we have sent out a survey and performed interviews. In the survey and interviews, we did not differentiate between the roles researchers can take in the academic publishing system. They were all readers and authors of academic publications; most were peer reviewers, and some were part of editorial teams of academic journals.\n\nIn the interviews, it became evident that trust plays an essential role in choosing a journal to publish in. Trust is based on various aspects: previous experiences with publishing and experiences with the journal, covering manuscript processing time, review quality, acceptance within the peer community, and some kind of measure representing the quality and trustworthiness of the journal.\n\nFor some scientists, the impact factor is the measure of quality and trustworthiness. Participant B8 mentioned that the journal’s impact factor is getting increasingly important due to the spread of predatory journals. The same interviewee also highlighted that prestigious journals with high impact factors, at the same time, are unreachable, so many authors do not even target their papers in these journals. So even though it is important for them to reach a wide audience, it is often more important to be realistic and aim for journals which would publish their papers with more certainty. “I am not sure if I ever published in a journal that had an impact factor larger than 10, to be honest. So these are also journals that I believe my more experienced colleagues fear, you know, because it is difficult to get in, right? Moreover, at the end of the day, you would like to, if you work for half a year on a publication, you would like to get it published. So they have very high criteria, and even if it is a good manuscript and an interesting topic, it might still not be as good as other submitted ones, right?”\n\nOthers mentioned that the impact factor is not the most important aspect when they choose where to publish, but it can happen that the chosen journals have, at the same time, a high citation index. “I find multidisciplinary journals and open access journals more interesting than five or six years ago. So, in the past, I liked a journal that I used to read very often. So, there were two or three journals that I knew were the articles that I was usually reading for my work. Then, these were usually very specific domain journals. And in these last years, I found more often that these open access multidisciplinarity journals publish much more interesting articles and they also have a higher citation index.” (Participant B5)\n\nManuscript processing time was also mentioned as an essential aspect when choosing a journal. Various interviewees (Participants B5 and B7) mentioned that when the review process of manuscripts is delayed, the authors are worried that their research findings would lose their newsworthiness or that they cannot add details of the already completed research when requested by the reviewers.\n\nTrust in open access\n\nAs open access is a widely used practice, we expected that researchers participating in our study would be aware of and have experience with publishing in such journals. To our surprise, the majority of the researchers not only knew about open access publishing but preferred open access to closed access. Two-thirds of the survey participants agreed or strongly agreed with “I believe free open access can be trusted”. At the same time, this number was 37% for the same statement about access behind a paywall. Researchers also stated that the open access process has a significantly higher level of integrity and demonstrates a significantly more benevolent attitude towards the scientific community when compared with the subscription-based model (Figure 2, Table 2 indicates the results of the Wilcoxon Signed Ranks test results).\n\nb Based on negative ranks.\n\nSeveral interviewees mentioned their moral concerns about the publishing system using paywalls and highlighted that they choose open access over closed publishing based on their values and beliefs. They mentioned that the profit-oriented model of publishers conflicted with the interests of the scientific community. “It is still strange to me that you give an article to a journal without getting paid, and they make money from it, while science should serve society and should not be behind some paywall.” (Participant A4) Another participant said that free access to all scientific publications in a given domain is a prerequisite to scientific integrity: “In my opinion, if I do not have access to all journals concerning my field, then I cannot provide integrity.” (Participant A1)\n\nSeveral interviewed researchers mentioned that the profit-oriented system blocks knowledge sharing within the universities (students do not have access to important current findings) and outside the walls of knowledge institutions, increasing the gap between science and society. One researcher mentioned that open access publishing allows sharing of the results with research participants (Participant A2). At the same time, another interviewee highlighted the importance of open access in science reaching decision-makers and having a broader impact by being implemented in policies (Participant B6).\n\nSeveral interviewees mentioned that their universities have an open access publishing policy (Interviewees A2 and B4), and the universities provide financial support to pay for the APC. Another participant mentioned that the grant provider subsidising their research project directed them to publish the research results in open access journals. However, for this latter participant, the compulsory nature of open access publication did not conflict with their values. “Even if I was able to publish, like, not Open access, I still think science should be free to everybody. Absolutely.” (Participant A1)\n\nIn the interviews, when asked about open access publishing, participants often mentioned other open science practices, such as sharing open code via GitHub, using open access data for their research and archiving data in repositories, often required by the grant institutions. In other cases, they reflected on the trend they experienced in the last years, moving towards more open science. “I have been in the business, so to say, for ten years; I have noticed that journals are becoming more and more willing to provide means to share open data with the public. For instance, via repositories and hyperlinks. Again, there is a trade-off. Because, as you might understand, becoming open also poses a threat in the sense that if everything is open and not well managed, all data becomes open.” (Participant B5)\n\nOne interviewee mentioned predatory journals taking advantage of scientists’ trust in open access journals. They mentioned that they try to avoid publishing in journals that directly approach them, and having selection criteria can help in falling prey to these journals. (Participant A2)\n\nPeer review\n\nIn general, researchers stated that they trusted the classical closed peer review system. They have found that this peer review system is competent in identifying errors or inconsistencies in papers, and they scored the integrity of this system high. It is noteworthy that the closed peer review system did not score high on predictability and benevolence (Figures 3 and 4).\n\nOne interviewee mentioned that the point of having a peer review is to have a discussion among peers to help the advancement of scientific knowledge, but the current system is selecting out those who disagree with the reviewers’ view. “I expect feedback from my peers because we want to build these ideas together. I am not a genius. And I do not think that knowledge is built by monolithic discourses or research; it is built together. To build that together, we definitely have to disagree on some points … But I think the peer review process now forgets a little bit about this, and it is more focused on how many publications I need to have per year, and people tend to look for the easy path there. They are like, avoid the person that is more critical with your approach.” (Participant B6)\n\nAs a significant critique, researchers mentioned in the interviews that they found the current closed peer review method too slow. “For example, an article I wrote two years ago is still not published. So I think that having more reviews is not always good. If you research very fast changing subjects, it can lead to frustrations.” (Participant A3) “Umm, so this is my personal frustration; it frustrates me quite quite a lot because I do realise I would like my results to be out there quicker than then they do now. “(Participant A1)\n\nIn the survey results, we found no significant difference between how participants trusted the closed and the public peer review options, except in one aspect: predictability. It seems that researchers trusted public peer review highly, but they would find this review method even more predictable than the closed one (Figure 3, Table 3).\n\nb Based on negative ranks.\n\nc Based on positive ranks.\n\nIn the interviews, participants highlighted the benefits of public review. Interviewee A1 acknowledged that they had experience only with a closed peer review system, which they trusted, but they found the idea of public peer review convincing and beneficial. “I think it is also good for the reviewers to know that at the end of the day, their names get out there, you know, for bad and for good. So I think if you know your name is going to be on something, you put more effort into it.” (Participant A1) Participant A4 could back this up; based on their experience, people are more constructive if they know that their peer review is published. Participant A2 highlighted the advantages of the public review method, especially for small, specialised scientific disciplines. They mentioned that they felt that the same people get the peer reviewer role when they want to publish, and they are asked to give peer reviews to the same people. “If the same person is always reviewing my work, they might just as well be co-authors. So I think maybe public review is better because then even the editorial team can look at who has reviewed previous work from the same author and decide; maybe we do not invite the same person over and over and over because we want different perspectives as well.” (Participant A2)\n\nResearchers reported that they trusted the participatory peer review significantly less than the public peer review system. They thought that a system that recruits peer reviewers from a broader scientific community would be less competent, predictable, benevolent, and have less integrity (Figure 4, Table 4). Participant A4 mentioned that the chance of finding someone knowledgeable in the field within the randomly assigned reviewers is probably low. Hence, the competence of these reviewers is probably lower, and therefore, the integrity of such a peer review system is also less.\n\nb Based on negative rank.\n\nGatekeeping\n\nPeople filling in the survey thought editorial teams are more competent, predictable, benevolent, and have more integrity than randomly assigned gatekeepers. From these aspects, the differences between trust, competency, and predictability were significant (Figure 5, Table 5).\n\nb Based on positive rank.\n\nWe gained some insights into why participants found the randomly assigned gatekeepers less trustworthy during the interviews. Participants A1 and A2 were concerned about the random gatekeepers’ experience, background and motivation, expecting that the random choice would not consider scientific domains and disciplines. Participant A1 mentioned that they were part of the editorial team of one journal at the time of the interview, representing the editorial’s perspective as well. “As a part of an editorial team, I would go for the first one [editorial board] because I think at least as a part of the editorial team of a journal, I do care for the journal. So, I try to act in the best interest of the journal when I approach it. From the randomly assigned people from the pure community, you might meet somebody interested, but you might as well meet somebody who is just doing it for the sake of putting another item on their CV. And they are just really not putting effort into the selection they make. So I would go for the first one.” (Participant A1)\n\nParticipant A1 also mentioned that a positive aspect of the current system is that authors can contact the editorial team and exchange ideas with them directly. Participant A2, although concerned about the experience of the randomly assigned gatekeepers, admitted that the randomly assigned gatekeeper system might be better because it can overcome favouritism if the gatekeepers come from discipline-specific communities. Participant A4 mentioned that this is not an easy question. They mentioned that editorial teams probably have more specific knowledge to make a solid decision based on their expertise. On the other hand, they highlighted that having the same editorial team for too long is not good. “If you let an editorial team sit for too long, you are more likely to get that kind of favouritism, and they will mainly pass on articles from people they know.” (Participant A4)\n\nGovernance\n\nResearchers trusted publishing companies with shared ownership policies more than publishers owned by private companies and investors. Researchers stated that those publishers are more predictable, benevolent, and competent, have more integrity, and have a shared governance model that includes the scientific community in decision-making. The differences between these two models were significant in all trust aspects (Figure 6, Table 6).\n\nb Based on negative ranks.\n\nDuring the interviews, one researcher highlighted the conflict between the concept of science as a community-driven endeavour and a single company-owned publisher, which defines which articles should be published. “I do not think it is appropriate for a scientific journal to belong to an individual or several individuals, mostly because I do not think science is a concept that goes with a company or private ownership.” (Participant A2) Another interviewee was more concerned about the profit-oriented nature of privately owned publishers: “For a company-owned structure, the more articles you accept, the more money you raise. And I think that is a dangerous trend”. (Participant A4). The same person mentioned that universities should play a much more critical role in publishing than they currently do.\n\nOne interviewee shared a potentially problematic issue related to shared ownership: reaching an agreement between the various perspectives and voices of the scientific community. “I think it would be complicated to combine all of these agendas and backgrounds and go towards a specific goal.” (Participant A4).\n\n\nDiscussion\n\nJournals and publishing platforms implementing various open science principles are considered alternatives to classical academic publishing. We were interested in figuring out to what extent Dutch researchers trust these new initiatives, especially given their concerns about predatory journals. We investigated various aspects of open science, namely open access, open peer review, random gatekeeping, and shared governance.\n\nBased on the literature review and our results, trustworthiness is the most important factor in choosing journals to read relevant scientific discoveries, and while it is also important in choosing journals to publish their papers, authors might have other priorities, too. Researchers at the beginning of their careers indicated more often that they rely on the impact factor of journals to define their trustworthiness, while experienced researchers based their decisions on where to publish on the familiarity of the journal, the acceptance of the journal within the peer community and their experience with the manuscript handling process and peer review services.\n\nMost researchers we interviewed mentioned that the quality of peer review of a given journal is crucial in deciding where to publish. This is in line with previous studies highlighting that authors might be willing to wait longer for better-quality feedback (Nickerson, 2005). This might contradict the authors’ wish to have a faster manuscript turnover time. Authors are afraid that their findings will lose their urgency and their results will become outdated or that they will lose track of the already finished and written study. Moreover, the reward system within academia pushes researchers to publish a certain number of papers within a set time frame (Bilalli, Munir, & Abelló, 2021). Some interviewees found it difficult to balance between the level of certainty of getting published in the short term and the journal’s reach. The trustworthiness of the journals could take a back seat in this decision.\n\nBased on previous research results, we expected to see a difference between early career researchers and tenured scientists because the institutionalisation process was suggested to fade the discontent with the current mainstream publication system (van Dijk & van Zelst, 2020). Nevertheless, we did not see any significant difference between the trust levels of researchers with various years of work experience.\n\nSome aspects of open publishing, such as open access, were well-known and widely accepted amongst the participants, while others, like participatory peer review or random gatekeeping, were less known. In these cases, many researchers formulated concerns about the competence of the randomly assigned gatekeeper and the reviewers coming from the wider community. Some of the researchers admitted that they did not have experiences with, e.g. open peer review, although they found this type of publishing benevolent and appreciated its added value. Some participants distrusted various aspects of open science because they found it hard to discriminate between serious open science publishers and predatory journals. This is a valid problem; multiple publications that draw attention to predatory journals do not discriminate between predatory papers and open access journals journals (Beall, 2013; Krawczyk & Kulczycki, 2021; Maurer et al., 2021; Torres, 2022).\n\nIn contrast to previous studies (Editage, 2018; Watkinson et al., 2016), researchers participating in this study trusted open access journals more than the ones using paywalls. When we asked why, they mainly claimed that profit-oriented publishers no longer represent the scientific community’s interests. Researchers listed various reasons why open-access publishing is important. The most frequently named one was that this way, students and non-scientific audiences could access these publications. Previous research showed that this is indeed the most commonly used argument for publishing in open access journals: increased audience size and increased impact of research findings (Taylor & Francis, 2019). Other arguments for open access mentioned in the interviews, such as the policies of the universities and funding organisations for publishing open access, also overlap with previous findings (Taylor & Francis, 2019). Other previous results were not confirmed or discussed by our study participants. Participants did not discuss receiving a higher number of citations by publishing in open-access journals (McKiernan et al., 2016) or retaining copyright via open access journals (compared to closed journals that demand the transferal of the rights over the published work (Watt & Sever, 2004).\n\nAlthough our participants trusted open access journals, several interviewees drew attention to the fast review and editing processes they implemented, which were not appreciated. The doubt of quality is one of the often-mentioned counterarguments against open access publishing found in literature as well, next to the inability or unwillingness to pay the APC, the claim that open access journals are not prestigious and finding the embargo policy of more prominent journals as an acceptable solution (Editage, 2018; Köster et al., 2021).\n\nOne participant mentioned the trend of open publishing in a broader sense, for example, publishing code and data, and also highlighted the importance of finding a balance between privacy and openness.\n\nThe participants of this study trusted the closed peer review system; they thought that it was a competent system for quality control. It also scored high on integrity, while its benevolence and predictability were not that much appreciated. Based on the questionnaire results, researchers trusted open-review journals as much as journals that used closed peer review. This contradicts previous research results. A study from 2008 showed that 80% of authors preferred double-blind, 52% single-blind closed peer review, compared to 27% of authors who would have chosen open peer review (Ware, 2008), but the acceptance of open review might have changed since 2008. Still, a small study from 2020 showed that researchers are hesitant to publish in journals with open peer review because, among others, they were afraid that reviewers would self-censor their reviews (Besançon, Rönnberg, Löwgren, Tennant, & Cooper, 2020). The survey results claimed that researchers would find open review journals more predictable, while in the interviews, other aspects were also mentioned, such as open peer review can lead to more constructive feedback and that it can advance transparency. These aspects were also mentioned in previous studies (Ross-Hellauer, Deppe, & Schmidt, 2017).\n\nCompared to the open and closed peer review options, participatory peer review was not found to be trustworthy. Participants thought that having peer reviews from the extended peer community would be less competent, predictable, benevolent, and have less integrity. Similarly, randomly assigned gatekeepers were also found to be less competent, predictable, benevolent, and have less integrity than editorial boards. Researchers were concerned about the experience, background and especially the motivations of these randomly selected gatekeepers.\n\nIn contrast with participatory peer review and randomly assigned gatekeepers, publishers with shared ownership were found to be more trustworthy than journals or publishing platforms owned by private companies. The relationship between closed publishing and company-owned publishers was highlighted and highly distrusted. The reasons behind the distrust were mostly moral, claiming that the concept of science does not fit private ownership and the profit orientation.\n\nIn this study, we investigated a limited set of publishing practices and excluded collaborative writing, open infrastructures, and preprints due to limited time and resources. Further studies are suggested to investigate these other aspects of Open Science.\n\nFurthermore, we included researchers working in Dutch Universities. This might limit the generalisation of the results.\n\n\nConclusion\n\nThe academic publication system is changing. Various scientists have formulated issues with classical publication methods, such as the generated scientific knowledge not reaching a wide enough audience, the inherent biases generated by governance and gatekeeping, or the publish-or-perish phenomenon, just to name a few. Open Science aims to provide various solutions for some of these problems.\n\nIn the academic publishing system, multiple actors are in (inter-)dependent relationships with each other. In almost all situations, there is a high level of uncertainty about whether the actors can reach their goals via the other interaction partners. Therefore, we can assume that trust relationships are present in the context of academic publishing.\n\nWe have created a theoretical framework to investigate trust in the context of academic publishing. Using competence, benevolence, integrity, and predictability to make the construct of trust more tangible, we could find nuances in which aspects of current and new publishing solutions are important for researchers and which ones are more trusted.\n\nBased on a questionnaire and twelve interviews, we saw that Dutch researchers trust and accept the new publishing concepts of open access, open peer review, and shared ownership in publishers. Participatory or extended peer review and randomly assigned gatekeepers instead of editorial boards are found to be less competent and, therefore, less trustworthy. These results indicate changes in the social norms within the Dutch scientific community, especially related to the profit-oriented nature of scientific publishing and the importance of open access to scientific results.\n\nBased on our results, we can draw a picture of an ideal publisher for the current Dutch researchers who would like to publish their scientific results. If one would create a new publishing platform based on the needs, social norms and values of the Dutch researchers, this publisher\n\n• should have a non-profit ownership model, controlled by a community of experts and/or governed by universities\n\n• should publish open access papers, preprints and peer reviews, with considerations on the publication size and format\n\n• should have some degree of gatekeeping, with a strong peer review process, ensuring quality control mechanisms of the reviews by potentially introducing meta-reviews and automation for speeding up the process\n\n• should keep a reputation-like score that rewards quality peer reviews and discourages the misuse of the system.\n\nThe results of our interviews also indicate that the successful implementation of Open Science practices requires a fundamental system change in the research life cycle because academic publishing is still an important part of assessing scientists’ excellence. We strongly believe that changes in the academic ecosystem are much needed. We also strongly believe that these changes need to be co-designed with all stakeholders to realise the transition from the current practices to Open Science. We need to take along scientists, but also publishers, librarians, decision-makers, societal stakeholders, patent and grant officers along the way to create the solutions together. Otherwise, situations could occur in which Open Science-based solutions are implemented while various stakeholders do not trust some aspects of these.", "appendix": "Data availability\n\nDANS: Trust in Open Publishing Practices – ideation session. https://doi.org/10.17026/SS/XX1LUW (Kalmar, 2024g).\n\nThis project contains the ideation session.pdf file, which describes the ideation session, the results collected during it and the analysis of the results. It is licensed under the CC_BY-NC-ND-4.0. The ideation session document does not contain any personal or sensitive information, but due to the agreement signed in the informed consent forms, which were reviewed and accepted by the TUD HREC committee, stating that the anonymised results can be shared by the responsible researcher upon request, access to the document is restricted. If you wish to access the transcripts, please get in touch with the corresponding author of this article via email: e.kalmar-1@tudelft.nl. Access will only be granted for research purposes, for research projects that are relevant to this topic.\n\nDANS: Trust in Open Publishing Practices - interview series B. https://doi.org/10.17026/SS/BUV2QO. (Kalmar, 2024c).\n\nThis project contains the interview transcript files (interview 1B.pdf, interview 2b.pdf, interview 3b.pdf, interview 4b.pdf, interview 5B.pdf, interview 6b.pdf, interview 7b.pdf and interview 8B.pdf ) under the license CC_BY-NC-ND-4.0. The interviews do not contain any personal or sensitive information, but due to the agreement signed in the informed consent forms, which were reviewed and accepted by the TUD HREC committee, stating that the anonymised transcripts can be shared by the responsible researcher upon request, access to the transcripts is restricted. If you wish to access the transcripts, please get in touch with the corresponding author of this article, via email: e.kalmar-1@tudelft.nl. Access will only be granted for research purposes, for research projects that are relevant to this topic.\n\nDANS: A Trust in Open Publishing Practices - interview series A. https://doi.org/10.17026/SS/P1XSH0 (Kalmar, 2024d).\n\nThis project contains the interview transcript files (interview 1 transcript.pdf, interview 2 transcript.pdf, interview 3 transcripts. pdf and interview 4 transcript.pdf ) under the license CC_BY-NC-ND-4.0. The interviews do not contain any personal information, but due to the agreement signed in the informed consent form, access to the transcripts is restricted. If you wish to access the transcripts, please get in touch with the corresponding author, of this article. Email: e.kalmar-1@tudelft.nl. Access will only be granted for research purposes, for research projects hat are relevant to this topic.\n\nDANS: Trust in Open Publishing Practices. https://doi.org/10.17026/SS/SOAFPP (Kalmar, 2024a).\n\nThe project contains the following underlying data:\n\n- survey data.ods\n\nDANS: Trust in Open Publishing Practices - questionnaire. https://doi.org/10.17026/SS/V51YDC (Kalmar, 2024b).\n\nThe project contains the following extended data:\n\n- survey.pdf\n\nDANS: Trust in Open Publishing Practices - interview protocol A, https://doi.org/10.17026/SS/TCB3ZD Kalmar, 2024e).\n\nThe project contains the following extended data:\n\n- interview protocol A.docx\n\nDANS: Trust in Open Publishing Practices - interview protocol B. https://doi.org/10.17026/SS/YAIGE5 (Kalmar, 2024f).\n\nThe project contains the following extended data:\n\n- Interview protocol B.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe guidelines from Standards for Reporting Qualitative Research (O’Brien, 2014) were applied in this report.\n\n\nReferences\n\nAmano T, Ramírez-Castañeda V, Berdejo-Espinola V, et al.: The manifold costs of being a non-native English speaker in science. PLoS Biology. 2023; 21(7): e3002184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBartling S, Fecher B: Blockchain for science and knowledge creation.2016. Publisher Full Text\n\nBartling S, Friesike S: Opening science: The evolving guide on how the internet is changing research, collaboration and scholarly publishing. Springer Nature; 2014. Reference Source\n\nBeall J: The open-access movement is not really about open access. tripleC: Communication, Capitalism, Critique. 2013; 11(2): 589–597. Publisher Full Text\n\nBesançon L, Rönnberg N, Löwgren J, et al.: Open up: a survey on open and non-anonymized peer reviewing. Research Integrity and Peer Review. 2020; 5: 1–11. Publisher Full Text\n\nBezuidenhout L, Chakauya E: Hidden concerns of sharing research data by low/middle-income country scientists. Global Bioethics. 2018; 29(1): 39–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBilalli B, Munir RF, Abelló A: A framework for assessing the peer review duration of journals: case study in computer science. Scientometrics. 2021; 126(1): 545–563. Publisher Full Text\n\nBorrego Á: Article processing charges for open access journal publishing: A review. Learned Publishing. 2023; 36(3): 359–378. Publisher Full Text\n\nBorycz J, Olendorf R, Specht A, et al.: Perceived benefits of open data are improving but scientists still lack resources, skills, and rewards. Humanities & Social Sciences Communications. 2023; 10(1): 1–12. Publisher Full Text\n\nBrembs B: Prestigious science journals struggle to reach even average reliability. Frontiers in Human Neuroscience. 2018; 12: 327726. Reference Source\n\nCallaway E: Scooped in science? Relax, credit will come your way. Nature. 2019; 575(7784): 576–577. PubMed Abstract \| Publisher Full Text\n\nCribb J, Sari T: Open science: sharing knowledge in the global century. Csiro Publishing; 2010.\n\nDavis GF, Cobb JA: Resource dependence theory: Past and future. emerald; 2010; vol. 28. .\n\nDietz G, Den Hartog DN: Measuring trust inside organisations. Personnel Review. 2006; 35(5): 557–588. Publisher Full Text\n\nEditage: 2018. AUTHOR PERSPECTIVES ON ACADEMIC PUBLISHING, GLOBAL SURVEY REPORT 2018.Reference Source\n\nEnagoAcademy: How to Suggest Reviewers for Your Paper.2021. Reference Source\n\nEshuis J, Van Woerkum C: Trust and monitoring in governance processes: lessons from landscape management by farmers in a Dutch municipality. Journal of Environmental Policy. 2003; 2003(4): 379–396.\n\nFerguson C, Marcus A, Oransky I: The peer-review scam. Nature. 2014; 515(7528): 480–482. PubMed Abstract \| Publisher Full Text\n\nFox CW, Meyer J, Aimé E: Double-blind peer review affects reviewer ratings and editor decisions at an ecology journal. Functional Ecology. 2023; 37(5): 1144–1157. Publisher Full Text\n\nFuchs C, Sandoval M: The diamond model of open access publishing: Why policy makers, scholars, universities, libraries, labour unions and the publishing world need to take non-commercial, non-profit open access serious. tripleC: Communication, Capitalism & Critique. 2013; 11(2): 428–443. Publisher Full Text\n\nGambetta D: Can we trust trust. Department of Sociology, University of Oxford; 2000; vol. 13. .\n\nGerwing TG, Allen Gerwing AM, Avery-Gomm S, et al.: Quantifying professionalism in peer review. Research Integrity and Peer Review. 2020; 5: 1–8. Publisher Full Text\n\nGomes DGE, Pottier P, Crystal-Ornelas R, et al.: Why don’t we share data and code? Perceived barriers and benefits to public archiving practices. Proceedings of the Royal Society B. 2022; 289(1987): 20221113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardwig J: The role of trust in knowledge. The Journal of Philosophy. 1991; 88(12): 693–708. Publisher Full Text\n\nHuang C-K, Neylon C, Montgomery L, et al.: Open access research outputs receive more diverse citations. Scientometrics. 2024; 1–21. Publisher Full Text\n\nHummels H, Roosendaal HE: Trust in scientific publishing. Journal of Business Ethics. 2001; 34: 87–100. Publisher Full Text\n\nJackson L, Peters MA, Benade L, et al.: Is peer review in academic publishing still working? The Methodology and Philosophy of Collective Writing. Routledge; 2021; pp. 154–172. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices [survey]. [dataset]. 2024a. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices - questionnaire. [dataset]. 2024b. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices - interview series B. [dataset]. 2024c. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices - interview series A. [dataset]. 2024d. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices - interview protocol A. [dataset]. 2024e. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices - interview protocol B. [dataset]. 2024f. Publisher Full Text\n\nKalmar E: Trust in Open Publishing Practices – ideation session. [dataset]. 2024g. Publisher Full Text\n\nKhadilkar SS: Rejection blues: why do research papers get rejected? The Journal of Obstetrics Gynecology of India. 2018; 68: 239–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan H, Almoli E, Franco MC, et al.: Open science failed to penetrate academic hiring practices: a cross-sectional study. Journal of Clinical Epidemiology. 2022; 144: 136–143. PubMed Abstract \| Publisher Full Text\n\nKlebel T, Ross-Hellauer T: The APC-barrier and its effect on stratification in open access publishing. Quantitative Science Studies. 2023; 4(1): 22–43. Publisher Full Text\n\nKöster M, Moors A, De Houwer J, et al.: Behavioral reluctance in adopting open access publishing: Insights from a goal-directed perspective. Frontiers in Psychology. 2021; 12: 649915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrawczyk F, Kulczycki E: How is open access accused of being predatory? The impact of Beall’s lists of predatory journals on academic publishing. The Journal of Academic Librarianship. 2021; 47(2): 102271. Publisher Full Text\n\nKulkarni S: What causes peer review scams and how can they be prevented? Learned Publishing. 2016; 29(3): 211–213. Publisher Full Text\n\nLanier WL: Dealing with inappropriate-, low-quality-, and other forms of challenging peer review, including hostile referees and inflammatory or confusing critiques: Prevention and treatment. Accountability in Research. 2021; 28(3): 162–185. Publisher Full Text\n\nLarivière V, Haustein S, Mongeon P: The oligopoly of academic publishers in the digital era. PLoS One. 2015; 10(6): e0127502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee I: Publish or perish: The myth and reality of academic publishing. Language Teaching. 2014; 47(2): 250–261. Publisher Full Text\n\nLiu F, Rahwan T, AlShebli B: Non-White scientists appear on fewer editorial boards, spend more time under review, and receive fewer citations. Proceedings of the National Academy of Sciences. 2023; 120(13): e2215324120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartín-Martín A, Orduna-Malea E, Thelwall M, et al.: Google Scholar, Web of Science, and Scopus: Which is best for me? Impact of Social Sciences Blog. 2019.\n\nMaurer E, Walter N, Histing T, et al.: Awareness of predatory journals and open access publishing among orthopaedic and trauma surgeons–results from an online survey in Germany. BMC Musculoskelet. Disord. 2021; 22: 1–8. Reference Source\n\nMcKiernan EC, Bourne PE, Brown CT, et al.: How open science helps researchers succeed. elife. 2016; 5: e16800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKiernan EC, Schimanski LA, Muñoz Nieves C, et al.: Use of the Journal Impact Factor in academic review, promotion, and tenure evaluations. elife. 2019; 8: e47338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorton B, Ranson H, El-Boghdadly K: How to measure academic impact. Anaesthesia. 2023; 78(5): 644–650. Publisher Full Text\n\nNabyonga-Orem J, Asamani JA, Nyirenda T, et al.: Article processing charges are stalling the progress of African researchers: a call for urgent reforms. BMJ Global Health. 2020; 5(9): e003650. Reference Source\n\nNakamura RK, Mann LS, Lindner MD, et al.: An experimental test of the effects of redacting grant applicant identifiers on peer review outcomes. elife. 2021; 10: e71368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNewhouse LJ, Brandeau ML: Who are the gatekeepers? An examination of diversity in Informs journal editorial boards. Serv. Sci. 2021; 13(3): 109–132. Publisher Full Text\n\nNeylon C, Huang C: The Global State of Open Access 2021.2022. Reference Source\n\nNicholas D, Watkinson A, Jamali HR, et al.: Peer review: Still king in the digital age. Learned Publishing. 2015; 28(1): 15–21. Publisher Full Text\n\nNickerson RS: What Authors Want From Journal Reviewers and Editors.2005.\n\nNiya SR, Pelloni L, Wullschleger S, et al.: A Blockchain-based Scientific Publishing Platform. Paper presented at the 2019 IEEE International Conference on Blockchain and Cryptocurrency (ICBC). 2019.\n\nNooteboom B, Six F: The Trust Process in Organizations: Empirical Studies of the Determinants and the Process of Trust Development. Edward Elgar Publishing; 2003. Publisher Full Text\n\nO’Brien BC, Harris IB, Beckman TJ, et al.: Standards for reporting qualitative research: a synthesis of recommendations. Academic Medicine. 2014; 89(9): 1245–1251.\n\nO’Hara K: A general definition of trust. Web & Internet Science. 2012.\n\nOmer A, Abdularhim M: The criteria of constructive feedback: The feedback that counts. Journal of Health Specialties. 2017; 5(1): 45–45. Publisher Full Text\n\nOnie S: Redesign open science for Asia, Africa and Latin America. Nature. 2020; 587(7832): 35–37. PubMed Abstract \| Publisher Full Text\n\nOwens SR: Revolution or evolution? A shift to an open-access model of publishing would clearly benefit science, but who should pay? EMBO Rep. 2003; 4(8): 741–743. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhelps L, Fox BA, Marincola FM: Supporting the advancement of science: Open access publishing and the role of mandates. Journal of Translational Medicine. 2012; 10(1): 1–3. Publisher Full Text\n\nPrimack RB, Regan TJ, Devictor V, et al.: Are scientific editors reliable gatekeepers of the publication process? Biological Conservation. 2019; 238: 108232. Publisher Full Text\n\nRavenscroft J, Liakata M, Clare A, et al.: Measuring scientific impact beyond academia: An assessment of existing impact metrics and proposed improvements. PLoS One. 2017; 12(3): e0173152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResnik DB, Elmore SA: Ensuring the quality, fairness, and integrity of journal peer review: A possible role of editors. Sci. Eng. Ethics. 2016; 22: 169–188. PubMed Abstract \| Publisher Full Text\n\nReynolds A: Reducing Bias in Scientific Publication through an Open Access Repository.2016.\n\nRoss-Hellauer T: What is open peer review? A systematic review. FResearch. 2017; 6: 588. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoss-Hellauer T, Deppe A, Schmidt B: Survey on open peer review: Attitudes and experience amongst editors, authors and reviewers. PLoS One. 2017; 12(12): e0189311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchimanski L, Alperin J: The evaluation of scholarship in academic promotion and tenure processes: Past, present, and future [version 1; peer review: 2 approved]. F1000Res. 2018; 7(1605): 1605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt B, Ross-Hellauer T, van Edig X , et al.: Ten considerations for open peer review. FResearch. 2018; 7. Publisher Full Text\n\nShrestha J: Predatory journals as threats to the academic publishing: a review. Journal of Agriculture and Natural Resources. 2021; 4(2): 1–10. Publisher Full Text\n\nStarfield S, Paltridge BJ: Journal editors: Gatekeepers or custodians?.Habibie P, Hyland K, editors. Novice Writers and Scholarly Publication. Palgrave Macmillan; 2019; pp. 253–270. Publisher Full Text\n\nStephan PE: The Economics of Science. J. Econ. Lit. 1996; 34(3): 1199–1235. Reference Source\n\nTaylor & Francis: Peer review in 2015: A global view. Taylor & Francis Group; 2015. Reference Source\n\nTaylor & Francis: Taylor & Francis Researcher Survey.2019. Reference Source\n\nTeng Y: Towards trustworthy blockchains: normative reflections on blockchain-enabled virtual institutions. Ethics Information Technology. 2021; 23(3): 385–397. Publisher Full Text\n\nTennant JP, Dugan JM, Graziotin D, et al.: A multi-disciplinary perspective on emergent and future innovations in peer review. FResearch. 2017; 6: 1151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTenopir C: Trust in reading, citing and publishing. Information Services & Use. 2014; 34(1-2): 39–48. Publisher Full Text\n\nTenorio-Fornés Á, Tirador EP, Sánchez-Ruiz AA, et al.: Decentralizing science: Towards an interoperable open peer review ecosystem using blockchain. Information Processing and Management. 2021; 58(6): 102724. Publisher Full Text\n\nTorres CG: Editorial misconduct: the case of online predatory journals. Heliyon. 2022; 8(3). Reference Source\n\nUNESCO: UNESCO Recommendation on Open Science.Reference Source\n\nVaught M, Jordan DC, Bastian H: Concern noted: a descriptive study of editorial expressions of concern in PubMed and PubMed Central. Research Integrity and Peer Review. 2017; 2: 1–11. Publisher Full Text\n\nvan Dijk H , van Zelst M : Comfortably numb? Researchers’ satisfaction with the publication system and a proposal for radical change. Publications. 2020; 8(1): 14. Publisher Full Text\n\nVan Noorden R: The science That’s never been published. Nature. 2017; 552: 162–164. PubMed Abstract \| Publisher Full Text\n\nvan Rossum J: Blockchain for research. Science. November 2017.\n\nWalker R, Rocha da Silva P: Emerging trends in peer review—a survey. Front. Neurosci. 2015; 9: 139226. Reference Source\n\nWare M: Peer review: benefits, perceptions and alternatives. Citeseer; 2008.\n\nWare M, Mabe M: The STM report: An overview of scientific and scholarly journal publishing.2015.\n\nWatkinson A, Nicholas D, Thornley C, et al.: Changes in the digital scholarly environment and issues of trust: An exploratory, qualitative analysis. Information Processing Management. 2016; 52(3): 446–458. Reference Source\n\nWatt FM, Sever R: Non-profit publishing: Open access and the end of copyright transfer. J. Cell Sci. 2004; 117(1): 1–1. PubMed Abstract \| Publisher Full Text\n\nWertz RJ: Saldaña J, editor. The coding manual for qualitative researchers.2014; 6(1): 128–130.\n\nWilliams AE: Altmetrics: an overview and evaluation. Online Information Review. 2017; 41(3): 311–317. Publisher Full Text\n\nWolfram D, Wang P, Hembree A, et al.: Open peer review: promoting transparency in open science. Scientometrics. 2020; 125(2): 1033–1051. Publisher Full Text" }	[ { "id": "310402", "date": "21 Aug 2024", "name": "Lex Bouter", "expertise": [ "Reviewer Expertise Scholar of research integrity and open science. Expert in epidemiological research methods", "also versed in survey methods", "qualitative research and biostatistics." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the results of a survey and a number of interviews among Dutch researchers on whether a number of innovative open publication practices are positively associated with the trustworthiness of journals. While this is an important and relevant question, the manuscript is at least twice too long and very difficult to follow due to many side paths, inconsistencies in the terminology and its opaque structure. It took me a while to figure it out, but I believe that the core concerns X innovative open publication practices and their association with Y aspects of trustworthiness. But this is buried under a huge set of side plots and lose ends. It also remains unclear how large X and Y are and how these concepts are defined and named. It’s important that these issues are repaired in the revision.\nRecommendations for the content and structure of the revised manuscript\nI believe that the core problem is that too much is piled in one manuscript. Please consider removing the desk research and the ideation session. Report these elements of your project elsewhere, e.g. as preprint to enable referencing these sources. The revised manuscript can then focus on the survey and the interviews. If you do this, the Introduction section can concentrate on introducing the open publication practices and aspects of trustworthiness you studied, and can make clear how many these are and which names you gave them throughout the article (one name per concept please). You can of course base these on the conclusions of the desk research and the ideation session, but should refer readers interested in how you arrived at these conclusions to the separate reports of these founding elements of your project. The Methods section can then also be shortened substantially and refer explicitly to the publicly accessible study protocols of the survey and the interviews, and the full sets of survey questions and interview scripts. Thus scholars who want to check or replicate your work can then find all details they need in the DANS repository, including the anonymized data sets. Please explain clearly how you operationalized the innovative publication practices and aspects of trustworthiness in your study, and make clear for each of them whether these were psychometrically validated. I believe that the Discussion section should be restructured and that the current Conclusion section should be integrated in it. Please summarize your main findings, list your study limitations, situate your findings in the context of what has ben published before, and lastly explain what they imply and what should happen next in terms of future research or implementation. Finally of course the abstract needs to be revised completely, sticking closely (including terminology) to the revised sections of the manuscript.\nOther recommendations\nExplain whether you preregistered your study. If so, provide the link to the preregistration. If not, explain why. You only report how many researchers participated in the survey and the interviews, but do not say how many were invited and how exactly you selected the invitees. Please provide response rates both for the survey and the interviews. In Figures 2, 3, 4, 5, and 6 you present contrasts between innovative open publication practices and presumably their traditional counterparts. Please make explicit what you wanted to do, how these traditional counterparts were defined and why you selected these comparators. Make clear whether the data analyses (of both the survey and the interviews) were done in duplicate and whether there was good agreement between these independent analyses. Please make very clear that you studied views of researchers on open publication practices and their associations with perceived trustworthiness at one single time point. Please explain explicitly that this does not enable conclusions about changes over time or about causality. The paragraph on study limitations is incomplete in my view. Examples of what I was missing: small sample sizes, low response rates (selective non-response), multiple testing (chance findings due to that), ad hoc in stead of validated measurement instruments (survey and interview questions), lack of attention to existing huge differences between journals, limited experiences of the participating researchers (especially early career researchers). Furthermore, it should be acknowledged that the study participants mainly came from one technical university, which is not representative for the Dutch academic landscape.\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": [] }, { "id": "334867", "date": "02 Dec 2024", "name": "Amanda Eiser Hess", "expertise": [ "Reviewer Expertise Special 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 for the opportunity to review “Trust in open publishing practices.” This paper has multiple strengths, including the use of various data collection methods, a commitment to open science practices, and empirically examining an important but under-studies issue (i.e., trust in open publishing practices). However, as detailed below, we have some concerns related to the clarity, methods, and overall structure of the manuscript that need to be addressed before we feel it should be considered for getting indexed into bibliographic databases.\nIntroduction:\nWe feel the paper would benefit from a clear and compelling conceptual framework. In what ways is trust lacking in traditional publishing? How might the specific open practices engender trust? We suggest moving the content on developing the framework regarding trust, which delineates the key elements of trust in relation to publishing, to the Introduction. Most broadly, we feel the authors could provide a stronger context for the study and make a more compelling case for its importance.\n\nMethod:\nWe suggest describing interview series A and B when, not after, they are introduced (p. 4). Explicitly state that this is a mixed-methods study. Was a particular type of mixed-method design applied?\n\nSampling concerns: There is no way of discerning response rate due to recruitment taking place on social media, which raises issues of sampling bias. We would appreciate -\n\n(a) more information on sampling and the sample (i.e., recruitment, inclusion/exclusion criteria [if any], sample characteristics) to assess representativeness of the sample and\n\n(b) a discussion of the threats to external validity of the survey due to these procedures (perhaps in the Limitations section).\n\nThe “desk research” section seems disconnected from the rest of the study. Consider removing this section and/or reporting findings in Introduction to provide context and a rationale for the study. The idea generation session appears to focus more on general problems in publishing, rather than trust specifically. On what basis did the authors conclude that trust was the main theme and should be the focus of the rest of the study? Was a formal approach/methodology used here? What techniques were used to enhance the credibility of findings? Survey: Where do the key areas of competence, predictability, benevolence, and integrity come from? It seems important to provide a strong justification for these key components of trust. What is an open science institution? Interviews: The interview samples (4 researchers from survey and 8 non-participants) appear neither purposeful nor representative. A more detailed rationale for sampling would strengthen this. The authors surveyed attitudes related to trust, integrity, and benevolence for open access publishing, but examined trust, competence, predictability, integrity, and benevolence for other practices. Consider providing a more detailed rationale for not assessing all constructs for open access and/or a rationale for examining all areas of trust for the other open practices. The definition of trust (“trust is an attitude that one takes to the trustworthiness of another; in turn, the other’s trustworthiness is a property that they have”) is unclear and appears circular to us. Please consider providing a more meaningful definition. Analysis: Please provide a description of specific statistical analyses conducted (rather than note “statistical analyses were performed”).\n\nResults:\nConsider providing a definition and description of random gatekeepers (p. 13). It would be helpful to introduce and define this and other key constructs in the Introduction section. The ideation session appears to not just be about openness. For example, having a rating or evaluation system for peer reviewers based on the quality of their feedback and integrating an automatic process in the peer review by implementing an AI-based pre-review are not related to openness. It was not clear to us how this content fits with rest of study. “Based on the categories that emerged during the idea selection stage of the ideation session, we have decided to focus on the following aspects of open publishing during the rest of the research: open access, peer review, gatekeeping, and governance.” Some basis for why some open practices were excluded, others included, and the focus on trust is needed. Did trust emerge as a critical constellation of concerns around publishing? Seems like more is needed here to describe the analytic procedures used to arrive at these conclusions (which should be described in the Method section). Alternatively, the authors might move this content to the Introduction and treat as context for study rationale. We suggest examining and reporting psychometrics (e.g., reliability) of survey. Integration of results: The connection between the study’s findings and trust constructs could be made more consistent and clearer.\n\nDiscussion\n“Based on the literature review and our results, trustworthiness is the most important factor in choosing journals to read relevant scientific discoveries” (p. 14). “Based on our results, we can draw a picture of an ideal publisher for the current Dutch researchers who would like to publish their scientific results” (p. 16). How were these conclusions reached? It is not clear to us that these claims are supported by study findings. Issues related to sample (size, neither representative [quantitative] nor purposive [qualitative]) should be included in the Limitations section (p. 16).\n\nIn summary, we felt that the paper addresses an important topic and has the potential to contribute to the literature base regarding scholars’ perceptions of trust related to open-publishing practices. However, as written, the paper jumps from broad issues in publishing, to open-science practices, to trust, and does not always provide a compelling and coherent conceptual framework for the study as a whole or connecting the different elements of the study. We also have concerns related the methods and the validity of the findings. If and when the authors can address these concerns, we believe that the article can be approved for indexing. That being said, it is unclear to us whether all of these issues can be addressed through a revision. We wish the authors well as they continue their work on this important topic and hope they found this review constructive.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-851
https://f1000research.com/articles/13-850/v1	30 Jul 24	{ "type": "Research Article", "title": "Unraveling the Interplay of Autophagy Genes and KLF3/KLF8 in Colorectal Cancer Metastasis: A Bioinformatics and cellular Exploration", "authors": [ "Eglal Mahgoub", "Jalal Taneera", "Samrein B. Ahmed", "shirin hafezi", "Thenmozhi Venkatachalam", "Mahmood Hachim", "Nabil Sulaiman", "Rifat Hamoudi", "Maha Saber-Ayad", "Jalal Taneera", "Samrein B. Ahmed", "shirin hafezi", "Thenmozhi Venkatachalam", "Mahmood Hachim", "Nabil Sulaiman", "Rifat Hamoudi", "Maha Saber-Ayad" ], "abstract": "Background Colorectal cancer (CRC) is a widespread malignancy globally, yet effective therapeutic approaches for advanced, metastatic, and chemo-resistant cases remain limited. In this study, we knocked out CRC cell line HCT 116 for two autophagy genes (ATG5 and ATG7), then we conducted a transcriptomic analysis on those isogenic cell lines. which revealed an upregulation of Krϋppel-like factor 3 (KLF3) expression, that was biologically validated.\n\nMethods In this study, we performed CRISPR/Cas9 gene editing on HCT 116 followed with transcriptomics analysis on HCT 116 KO cells for ATG5 and ATG7. Various bioinformatics analyses were performed to investigate the KLF3/8 with autophagy and affected functional pathways, and immune genes related to the different types. Validation of expression in different cell lines were done using qPCR and Western blot.\n\nResults To further investigate the role of autophagy genes in CRC, we utilized publicly available data and web-based tools. Our analysis showed a marked correlation between KLF3/KLF8 and the expression of autophagy genes in CRC, denoting that its upregulation is likely to be a compensatory mechanism. We also examined the co-expression of autophagy genes and KLF3/KLF8 with multiple markers of epithelial-to-mesenchymal transition (EMT), and significant positive correlations were observed. Moreover, KLF8 expression was upregulated at the mRNA level in the metastatic cell lines LoVo and SK-CO-1, compared to HCT 116. Interestingly, KLF3/KLF8 expression was high in MSS molecular subtype of CRC as shown in HCT 116 cell line knocked in with MLH gene as well as they were negatively correlated with crucial immune-infiltrating cells such as CD8+ cells, indicating their potential as a negative biomarker for response to immunotherapy.\n\nConclusion Our study proposes that a synergistic approach involving the inhibition of KLF8 and autophagy holds a potential therapeutic target for effectively tackling metastatic CRC cells, especially in cases characterized by deficient mismatch repair (MMR).", "keywords": [ "Colorectal Cancer (CRC)", "Metastasis", "tumor progression", "Autophagy", "Krϋpple-like factor 8 (KLF8)", "MMR-Deficient", "chemoresistance." ], "content": "Introduction\n\nColorectal cancer (CRC) is a prevalent and deadly tumor affecting both men and women. It ranks second among the leading causes of cancer-related deaths worldwide, accounting for 9.2% of all such deaths, and is the third most prevalent cancer, representing 6.1% of new cases.1 Projections indicate a worrisome rise in deaths from colon and rectal cancers, expected to increase by 71.5% and 60%, respectively, by 2035.2 CRC is a complex disease with diverse clinical manifestations, molecular indicators, and prognosis.\n\nAutophagy, a cellular process, plays a significant role in the development of CRC and renders cancer cells less susceptible to chemotherapy. By promoting autophagy, cancer cells gain energy and essential metabolites.3 Autophagy ensures cellular homeostasis by facilitating the turnover of defective proteins and organelles, and its activation increases under cellular stress and nutritional scarcity.4–6 Autophagy may act as a tumor suppressor in the early stages of tumor formation, but it appears to promote tumor growth once tumors have already developed. Moreover, autophagy helps tumor cells overcome metabolic stress caused by rapid growth, hypoxia, and limited nutrient supply, which are characteristic of malignant tumors.7,8 Particularly under hypoxic and metabolic stress conditions, autophagy provides additional metabolites and energy to cancer cells.9,10 Importantly, autophagy has also been implicated in cancer cell metastasis, with Epithelial-Mesenchymal Transition (EMT) being a critical process for cancer cell invasiveness and metastasis.11 Regulation of autophagy is mediated via several transcription factors, or a transcriptional gene network.12 Add that autophagy inhibition did not show a marked suppressing effect on cancer cell proliferation, however, compelling evidence supports the notion that autophagy actively contributes to the migration and invasion of cancer cells.13\n\nRecent evidence suggests that Krüppel-like factors (KLFs) are key players in tumor development, growth, and metastasis. Several KLF members, including KLF4, KLF5, and KLF11, have been associated with the oncogenesis of various human cancers. KLFs belong to a family of zinc finger-containing transcription factors closely related to the general transcription factor Sp1. With over 26 known members, this family is characterized by a conserved C-terminus comprising three zinc finger DNA-binding domains. Dysregulation of several KLF family members has been observed in different human cancers, where they can function as either tumor suppressors or oncogenes, depending on the unique cellular context of cancer.14 Furthermore, KLFs are involved in various cellular processes, including differentiation, cell death, and cellular proliferation,15 modulation of autophagy and longevity (in C. elegans), with a potential role in vascular aging in mammals.16\n\nKrüppel-like factor 8 (KLF8) has been the focus of recent studies due to its role in regulating genes associated with various biological functions and pathological processes, such as proliferation, migration, invasion, and inflammation. Widespread expression of KLF8 has been observed in several cancer types, where it has been found to be essential for DNA repair and resistance to apoptosis.17–21 In gastric cell formation and progression, downregulation of KLF8 expression has been shown to decrease cell proliferation, migration, and invasion by reducing the expression of key factors involved in these processes.22\n\nIn this study, we utilized CRISPR/Cas9 to target ATG5 and ATG7; both are key genes involved in the autophagy machinery. Transcriptomics analysis revealed differential expression of the KLF3 transcription factor. It is worth noting that KLF8 features a distinctive DNA-binding domain at its C-terminus composed of three zinc fingers. Still, its N-terminal region also shares similarities with KLF3. Most significantly, it has a Pro-Val-Asp-Leu-Ser/Thr motif, shared with KLF3.23–25 Since KLF8 and KLF3 appeared to be co-expressed in multiple tissues and bind the identical DNA sequences and the protein partner CtBP, this suggests a potential functional overlap between these two transcription factors. However, the interplay between autophagy and KLF3/KLF8-mediated malignant phenotypes in CRC remains poorly characterized. Interestingly, autophagy is required for the degradation of KLF3, which might explain the high deferential expression of KLF3 in the autophagy knockout cell lines.26 Additionally, the association of autophagy with KLF family members, particularly group 1 (including KLF3 and KLF8), has not been previously investigated in tumors and may represent a novel therapeutic approach in CRC.\n\nHence, the primary objective of this study is to elucidate the roles of various autophagy-related genes and the involvement of KLF3/KLF8 in CRC carcinogenesis, focusing on evaluating their potential as prognostic markers and therapeutic targets for CRC.\n\n\nMethods\n\nA diverse panel of cell lines representing various stages of colorectal cancer was utilized. The panel included the HCT 116 cell line as the parental cell line and LoVo and SK-CO-1 as metastatic cell lines. HCT 116 (CCL-247), LoVo (CCL-229) and SK-CO-1 (HTB-39) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Additionally, two panels of cell lines were employed, consisting of the parent HCT 116 cell line (MLH1−/−) (RRID: CVCL_0291) and its isogenic counterpart, HCT 116 MLH1+/− (RRID: CVCL_HD84), were obtained from Horizon Discovery, gene editing company (Cat. No. HD104-006, Waterbeach, UK). The HCT 116 MLH1+/− cell line was obtained through CRISPR-Cas9 gene editing, where a single allele of the MLH1 gene was inserted. All cell lines were cultured under standard conditions at 37 °C in a humidified atmosphere with 95% air and 5% CO2. Specifically, the CRC cell lines were maintained in RPMI culture media (Sigma-Aldrich, Cat # R8758, Germany).\n\n1. CRISPR/Cas-9 gene editing\n\nWe used the ribonucleotide protein27 delivery system for CRISPR technique to knock out autophagy-related gene 5 (ATG-5) and ATG-7 in the HCT 116 cells. All the reagents were purchased from Thermo scientific company, USA. Two guide RNA (gRNA) were individually used to target ATG5/7 genes. Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent was used to transfect the cells with the gRNA and Cas9. The genomic cleavage efficiency was assessed by immunoblotting against the desired genes. TrueGuide sgRNA Positive Control, CDK428 was used in all steps as a positive control, while a scrambled gRNA, Trueguide gRNA Negative control, was used as a negative control in all the steps to ensure the specificity of the target. The pool of cells with the highest transfection efficiency was considered for the downstream validation. To generate isolated clones with a complete knockout, the mixed pool of cells was diluted 1 cell/200 μl liquid medium and then dispersed into 96 wells plates. The individual cells were left to form clones. The clones were tested for the expression of ATGF5/7 via immunoblotting.\n\nWhole transcriptome RNA sequencing was carried out on the HCT 116 control and ATG5 and ATG7 knockout using CRISPR/Cas9 system. About 100ng of total RNA was used for library preparation using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Scientific). About 100 pM purified library was taken and pooled equally with four individual samples per pool and were amplified using emulsion PCR on Ion OneTouch 2 instrument (OT2) (Thermo Fisher, RRID:SCR_023289, Cat # 4474778, USA) followed by enrichment using Ion OneTouch ES. Thus, prepared template libraries were then sequenced with Ion S5 XL Semiconductor sequencer using the Ion 540 Chip (Life Technologies, Cat # A27765, California, USA).\n\nRNA-seq data were analyzed using Ion Torrent Software Suite version 5.4 (Thermo Fisher, RRID:SCR_023289, Cat # 0017145, USA) and the alignment was carried out using the Torrent Mapping Alignment Program (TMAP).29 TMAP is optimized for aligning the raw sequencing reads against reference sequence derived from hg19 (GRCh37) assembly and the specificity and sensitivity were maintained by implementing a two-stage mapping approach by employing BWA-short, BWA-long, SSAHA,30 Super-maximal Exact Matching31 and Smith-Waterman algorithm32 for optimal mapping. Raw read counts of the targeted genes were performed using Samtools (Samtools view –c –F 4 –L bed_file bam_file) and the number of expressed transcripts was confirmed after Fragments Per Kilobase Million (FPKM) normalization.29\n\nA script was used (written in R programming language) was used to analyze the differentially expressed genes,10 with function calls to DESeq2 package from Bioconductor library sets. Raw read counts from RNASeq were normalized using quantile normalization. All counts ranked “0” were excluded. Differentially expressed genes between every two set of cell lines [ATG5-KO vs wild HCT 116 (NC), and ATG7-KO, vs wild HCT 116 (NC)] were assessed using 2 tailed t-test. Differentially expressed genes with p-value of <0.01, adjusted p-value <0.5 were included for analysis of pathways and transcription factors using Enrichr.\n\nRIPA lysis buffer (Cat. No. ab156034, Abcam) was used for protein extraction (30-80 μl according to pellet size), followed by centrifugation at 14,000 RPM for 15 min at 4°C. The protein concentration in the cell lysate was measured using a BCA kit (Cat. No. 23225, Thermo Scientific Pierce following the manufacturer’s instructions (Cat. No. 23227, Thermo Scientific Pierce, Waltham, MA, USA). A volume equivalent to 35μg of total protein was separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that was then electrophoretically transferred to PVDF membrane (Bio-Rad, USA). The membrane was blocked for one hour at room temperature using 5% Bovine Serum Albumin (BSA) powder (Sigma Aldrich).\n\nFollowing blocking, the membrane was washed with Tris-buffered saline with 0.1% Tween® 20 Detergent (TBST) and incubated overnight at 4 °C with primary antibodies, including anti-ATG5 (Cat. No. 12994S), anti-ATG7 (Cat. No. 8558S), anti-β-actin (Cat. No. 4970S) (all from Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were incubated with the membrane for 1 hour at room temperature at a ratio of 1:1000. The ECL kit (Thermo Scientific Pierce, Waltham, MA, USA) was used to identify chemiluminescence to identify protein bands, Bio-Rad Image Lab software (www.bio-rad.com, ChemiDocTM Touch Gel and Western Blot Imaging System; Bio-Rad, Hercules, CA, USA) was used.\n\nTotal RNA was extracted from cancer cells using Qiagen RNA Mini Kit (Cat. No. 12183018A, Thermo Scientific). The RNA quality and quantity were measured by a nanodrop2000 spectrophotometer (Thermo Scientific, USA). cDNA was synthesized from RNA using SensiFAST™ cDNA Synthesis Kit (Bioline Reagents Ltd., London, UK). Real-Time PCR was done for 2 genes encoding KLF8 and KLF3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. Quant Studio 3 (Thermo Fisher) was used (using SensiFAST™ SYBR Hi-ROX kit). The experiment was done in triplicates. Each reaction will yield the average threshold cycle (Ct) values for the genes, and the 2(−ΔΔC(T)) relative approach was used to quantify the expression (Table 1).\n\nTIMER2\n\nThe TIMER 2 web application (http://timer.cistrome.org) analyzes the co-expression of autophagy genes and KLF3/KLF8 in COAD and their expression with multiple EMT markers. TIMER 2 analyzes and quantifies the tumor-infiltrating immune cells from the TCGA dataset using a variety of algorithms. This instrument assesses the relationship between the abundance of immune-infiltrating cells and the gene of interest’s transcript. With the aid of this instrument, we looked into the relationship between autophagy genes and KLF3/KLF8 in CRC together and with different EMT markers.33 When choosing the TIMER algorithm and Spearman correlation, we first picked immune association to each gene and each type of immune cell.\n\nGene Expression Database of Normal and Tumor Tissues 2 (GENT2)\n\nWe used the GENT2 database http://gent2.appex.kr. to assess the expression of KLF3/KLF8 in various CRC subtypes.34 Microarray platforms (Affymetrix U133A or U133Plus2) are used in this tool’s NCBI GEO library.35 We used this tool to look into the expression of KLF3/KLF8 in CRC molecular subtypes, querying each gene individually across the molecular subtypes.\n\nGene Expression Profiling Interactive Analysis2 (GEPIA2)\n\nTo measure the autophagy genes and KLF3/KLF8 expression levels in different CRC stages, we used GEPIA2 web-based server. The data originates from the TCGA RNA Sequence Dataset (http://gepia2.cancer pku.cn/#index).36 By looking up each gene under the stages tab individually.\n\nUALCAN\n\nUALCAN is a web-based tool (http://ualcan.path.uab.edu) that utilizes OMICS data from cancer data sets like TCGA, MET500, and CPTAC. Studying the promotor methylation level in different stages of CRC and in different stages of lymph node metastasis and determining significance using the Student t-test between two applications.37 The Beta value indicates level of DNA methylation ranging from 0 (unmethylated) to 1 (fully methylated). Different beta value cut-off has been considered to indicate hyper-methylation [Beta value: 0.7-0.5] or hypo-methylation [Beta-value: 0.3-0.25].\n\nGene Set Cancer Analysis (GSCALite), a web-based server, is used to look into the state of genes in datasets from cancer patients (http://bioinfo.life.hust.edu.cn/web/GSCALite/).38 Interestingly, we used this tool to examine the function of autophagy genes and KLF3/KLF8 in cancer-related pathways in the COAD data set from TCGA, as it can predict gene activity in cancer-associated pathways.\n\nThe GeneMANIA database (http://genemania.org)39 was utilized to generate hypothesis regarding the interactions between KLF3/KLF8 with a total of twenty proteins that surround it. Protein-protein interactions were examined based on several networks, including as co-expression and common pathways, using KLF3/KLF8 as a research tool.\n\nThe data were analyzed using various statistical tests, as described in the figure legends. The mean values are presented from at least three independent experiments. The differences between different cell lines in KLF3 and KLF8 were assessed using the Student’s T-test for the qPCR experiments. All statistical analyses were performed using GraphPad Prism software (version 8.0.0 www.graphpad.com) (San Diego, CA, USA). P-values less than 0.05 were considered significant.\n\n\nResults\n\nWe evaluated the expression of ATG5/7 in multiple CRC cell lines in which we found high expression levels in HCT 116, LoVo and SK-CO-1 while HT29 cell line showed relatively low expression (Figure 1A). However, previous literature showed that HCT 116 showed a higher controlled autophagy flux compared to other CRC cell lines.40 Thus, HCT 116 has been used to knockout ATG5/7 via CRISPR/Cas9 editing techniques.\n\n(A) Representative Western blot of ATG5, ATG7 levels in a panel of CRC cell lines including: HCT 116, HT29, LoVo, SK-CO-1. β-actin was used as the loading control.\n\n(B) Representative Western blot of ATG5 and ATG7 Knocking out of the HCT116 cell line using CRISPR/Cas9 gene editing. Colony selection was carried out to ensure the purity of the cell population knocked out for ATG5 and ATG7.\n\n(C) Whole transcriptomic analysis in ATG5 KO cell lines captured multiple differentially upregulated pathways.\n\n(D) Whole transcriptomic analysis in ATG7 KO cell lines captured multiple differentially upregulated pathways.\n\n(E) Lower panel shows top differentially expressed transcription factors predicted to be upregulated in HCT 116 KO for ATG5.\n\n(F) Lower panel shows top differentially expressed transcription factors predicted to be upregulated in HCT 116 KO for ATG7.\n\n(G) The Venn chart shows the shared genes among top differentially upregulated pathways in both ATG5-KO and ATG7- KO HCT 116 cell lines, showing KLF3 transcriptional factor as the top upregulated gene in both.\n\nThe GSEA relies on protein-protein interactions for transcription factors extracted from several literature databases (Transcription Factor PPI). The analysis was performed using Enricher web tool. The Gene Ontology (GO) pathways were ranked according to the lowest adjusted p-value representing statistical significance between the KO cell line and NC.\n\nWe applied CRISPR/Cas9 gene editing to target ATG5 and ATG7 to get HCT 116-knockout ATG5 and HCT 116-knockout ATG7 (Figure 1B).41 Whole transcriptomic analysis in those KO cell lines captured multiple differentially upregulated pathways in ATG5 KO and ATG7 KO as shown in Figure 1C, 1D, respectively. The top shared genes among the upregulated pathways are shown in Figure 1E, 1F. Remarkably, KLF3 transcriptional factor was found to be the top upregulated gene in both KO cell lines (Figure 1G).41\n\nWe utilized GEPIA2 to investigate the expression levels of KLF3/KLF8 in different stages of colorectal cancer (CRC). As illustrated in Figure 2A, KLF3 expression was significantly increased in stages II and III compared to stage I. KLF8 expression significantly increased in the late tumor stages specifically stage IV (Figure 2B). Subsequently, we examined the methylation status of the promoter regions of KLF3 and KLF8 genes in various stages of CRC tumors and different stages of nodal metastasis using the UALCAN web tool. Our analysis revealed that the methylation level of the KLF3 promoter was significantly decreased in stage 4 of CRC tumors (Figure 2C). It showed significant change across N0 and N2 stages of lymph node invasion (Figure 2D). The methylation level of the KLF8 promoter was significantly decreased in stage 4 of CRC tumors and in late stages of lymph node invasion (Figure 2E, F). Overall, these findings suggest that KLF3 and KLF8 are associated with the progression of CRC, specifically in the later tumor stages.\n\n(A) KLF3 expression level in CRC stages: using GEPIA2 web server that provide RNA-seq from TCGA data set. (B) KLF8 expression level in CRC stages: using GEPIA2 web server that provide RNA-seq from TCGA data set. Y-axis represents the expression level of each gene as (log2(TPM+1)).TPM: Transcript per million.\n\n(C) KLF3 promotor methylation level in different stages COAD, (D) KLF8 promotor methylation level in different stages COAD, (E) KLF3 promotor methylation level in different stages of the lymph node metastasis, (F) KLF8 promotor methylation level in different stages of the lymph node metastasis. This is done using UALCAN web tool.\n\n(G) KLF3 gene expression comparison in different CRC cell lines: HCT 116, LoVo and SK-CO-1, (H) KLF8 gene expression comparison in different CRC cell lines: HCT 116, LoVo and SK-CO-1. Gene expression was analyzed using the Comparative Ct (ΔΔCt). Results are presented as mean (± SEM) relative to unstimulated control. The values were compared across the different groups using ANOVA test. ∗∗p < 0.01, ∗∗∗p < 0.001.\n\nTo validate the expression of KLF3 and KLF8 in CRC cells specifically in different tumor stages, we utilized metastatic cell lines LoVo and SK-CO-1 to detect KLF8 and KLF3 expression compared to the parental HCT 116 cell line via qPCR. We found a significant increase in the expression of KLF3 and KLF8 in both cell lines, compared to the HCT 116 cell line (Figure 2G, H).\n\nTo further investigate the relationship between autophagy genes and KLF3/KLF8 in colorectal cancer (CRC), we employed the TIMER2 web tool. Given our observation of distinct expression patterns of KLF3 in ATG5-/- and ATG7-/- knockout cells, our objective was to investigate the possible correlation and co-expression between autophagy genes and KLF3/KLF8 in CRC. The results, shown in Figures 3A, revealed significant positive correlations between KLF8 and majority of the autophagy genes, including ULK1, ULK2, beclin1, LC3B, ATG5, ATG7, AMBRA1, and most notably, UV radiation resistance associated gene (UVRAG) in both colon adenocarcinoma (COAD) (Figure 3A).41 Similarly, KLF3 exhibited significant positive correlations with ULK1, ULK2, BECN1, MAPLC3B, ATG5, ATG7, AMBRA1, and UVRAG in COAD (Figure 3B). These findings suggest a strong correlation between KLF3/KLF8 and the expression of autophagy genes in CRC.\n\nSpearman’s rank correlation coefficient obtained from TIMER 2 web tool of (A) KLF3 and autophagy-related genes co-expression in COAD, (B) KLF8 and autophagy-related genes co-expression in COAD.\n\nCo-expression of autophagy genes and EMT genes\n\nPrevious studies have indicated that autophagy promotes tumor progression and overcoming metabolic stress caused by rapid proliferation, hypoxia, and limited nutrient supply.7,8 Moreover, autophagy has implications in the multistep process of epithelial-to-mesenchymal transition (EMT).42,43 Therefore, we utilized the TIMER2 web tool to explore the potential relationships and co-expression patterns between autophagy genes, KLF3/KLF8, and EMT-related genes such as CDH1, CDH2, CTNNB1, SNAI1, SNAI2, VIM, and ZEB1, which represent E-cadherin, N-cadherin, Beta-Catenin, SNAIL1, SLUG, Vimentin, and ZEB1 markers, respectively.\n\nAs depicted in Figure 4, both ULK1 and ULK2 exhibited significant positive correlations with Beta-Catenin, SNAIL1, SLUG, Vimentin, and ZEB1 and showed relatively lower positive correlations with E-cadherin. Furthermore, beclin1 and LC3B were significantly positively correlated with ZEB1, Beta-Catenin, and SNAIL1. ATG7 and ATG5 showed significant positive correlations with ZEB1, Beta-Catenin, SLUG, Vimentin, and E-cadherin, while ATG10 exhibited a significant positive correlation with Beta-Catenin alone. Notably, UVRAG displayed substantial positive correlations with Beta-Catenin, SLUG, Vimentin, SNAIL1, and notably the highest positive correlation with the crucial EMT marker, ZEB1. These findings align with the idea that autophagy strongly correlates with the EMT process in CRC metastasis. Importantly, UVRAG, which exhibits the most significant expression correlation with KLF8, also displays the highest positive correlation with the key EMT marker, ZEB1. This indicates a substantial correlation between UVRAG, KLF8, and the EMT process.\n\nSimilarly, significant correlations were observed between KLF3, KLF8, and multiple EMT genes. As depicted in Figure 5A, KLF3 exhibited a significant positive correlation with Beta-catenin and ZEB1. At the same time, KLF8 displayed significant positive correlations with Beta-catenin, SLUG, N-cadherin, Vimentin, and notably, the highest positive correlation was observed with ZEB1 (Figure 5B). Interestingly, KLF8 was negatively associated with CDH1 gene coding for E-cadherin. These findings suggest that both autophagy and the Krüppel-like family, including KLF3 and KLF8, play a significant role in the Epithelial-Mesenchymal Transition (EMT) process in colorectal cancer (CRC). The correlations between these genes and EMT-related markers provide further evidence of their involvement in the EMT pathway, which is crucial for CRC metastasis and progression.\n\n(A) Spearman’s rank correlation coefficient obtained from TIMER 2 web tool of KLF3 and EMT markers co-expression in COAD, (B) Spearman’s rank correlation coefficient obtained from TIMER 2 web tool of KLF8 and EMT markers co-expression in COAD, (C) KLF8-protein interaction using STRING, (D) Shared protein domains associated with KLF3 using GeneMANIA, (E) Shared protein domains associated with KLF8 using GeneMANIA.\n\nSimilarly, using the web-based tool STRING, we performed 8-protein interactions. Interestingly, we found that KLF8 expression is positively associated with multiple EMT markers, including zeb1 and snai1 (Snail) (Figure 5C). Moreover, Using GeneMANIA, a protein-protein interaction network further confirmed the direct correlation of KLF3 and KLF8 EMT markers as illustrated in Figure 5D and 5E, respectively.\n\nAfter investigating the status of KLF3/KLF8 co-expression with autophagy genes as well as EMT markers, we utilized GSCALite to determine the molecular functions of these genes in COAD (Figure 6A, B) and across all cancer types (Figure 6C, D). We found significant correlations in COAD between specific autophagy genes and essential molecular functions. The AMBRA1 gene showed a significant correlation with apoptosis inhibition. ULK2 and UVRAG genes were significantly correlated with activating the epithelial-mesenchymal transition (EMT). ATG7 gene displayed a significant correlation with activation of the RAS-MAPK pathway. Interestingly, ATG5 was correlated with EMT inhibition. Furthermore, in other cancer-related pathways across all cancer types, UVRAG exhibited positive correlations with the activation of DNA damage, estrogen hormone pathway, and receptor tyrosine kinase (RTK) pathway. These results, based on gene correlations with major regulators of cancer-related pathways, suggest that autophagy genes may play important roles in multiple cancer-related pathways.\n\n(A) COAD's predicted pathways of autophagy genes, (B) COAD's predicted pathways of KLF8, (C) Predicted pathways of autophagy genes in cancers, (D) Predicted pathways of KLF3/8 in cancers The color scale represents the percentage of activation and inhibition of different pathways in colorectal cancer, (E) Pathway-related genes associated with KLF8 using GeneMANIA.\n\nIn COAD, KLF8 gene exhibited significant correlations with apoptosis inhibition, cell cycle activation and EMT activation. Both KLF3 and KLF8 genes showed correlations with several cancer-related pathways. Specifically, KLF8 gene displayed positive correlations with the PI3-AKT, RAS-MAPK, and RTK pathways. Moreover, using GeneMANIA web tool (Figure 6E), pathways interactions in KLF8 showed enrichment of translational initiation of genes related to cell-cycle progression such as CDK4 and CDK6. Further, KLF8 was associated with CCND1which involved in promoting cell-cycle, invasion and metastasis.\n\nThese findings shed light on the molecular functions of autophagy genes and KLF3/KLF8 in COAD and across various cancer types. The correlations observed provide insights into the potential involvement of these genes in cancer-related pathways, suggesting their roles in key biological processes and signaling pathways associated with cancer development and progression.\n\nCRC is divided into key pathogenic molecular subtypes. The first group is microsatellite instable (MSI) due to mismatch repair (MMR) gene abnormalities (16%) or DNA polymerase epsilon proof reading (3%). The second subtype is microsatellite stable (MSS), which accounts for 84% of CRC cases. MSS CRC is distinguished by a high rate of somatic mutations in multiple genes, including APC, TP53, PIK3CA, KRAS, and SMAD4. We used the web-based program GENT2 to investigate the gene expression of KLF3/KLF8 in various tumor subtypes (Figure 7A, B). Comparing MSI and MSS subtypes, we found that KLF3 showed relatively high expression in MSS molecular subtypes of CRC, whereas KLF8 showed significantly high in MSS.\n\n(A) KLF3 expression level in colon cancer molecular subtypes, (B) KLF8 expression level in colon cancer molecular subtypes. Using GENT2 web- based tool to denote data from NCBI GEO database, we enquire COAD and choose subtype as the targeted analysis. Two sample T tests were conducted by the GENT2 web tool, p < 0.005 was considered as significant. (C) KLF8 gene expression in HCT116 and its isogenic cell line HCT116 MLH+/-, (D) KLF3 gene expression in HCT116 and its isogenic cell line HCT116 MLH+/-. The values were compared across the different groups using unpaired t test. ∗∗p < 0.01. Results are presented as mean (± SEM) of mRNA expression.\n\nMoreover, we included an isogenic cell line HCT 116 MLH1+/− with its parental HCT 116 cell line (MLH1−/−). We observed that MLH1 proficient cells were associated with high KLF8 expression but decreased KLF3 expression, compared to the wild-type MLH1 deficient (Figure 7C, D).\n\nWe conducted bioinformatics analysis using TIMER2 to assess the correlation between the expression of KLF3 and KLF8 genes and the cellular composition of the tumor microenvironment.44\n\nKLF3 displayed significant positive correlations with CD8+ T cells, CD4+ T cells, neutrophils, Tregs, and NK cells. However, a significant negative correlation existed between KLF3 expression and M2 MCQ infiltration. KLF3 showed no significant correlation with MDSC (Figure 8A). Similarly, KLF8 exhibited significant positive correlations with CD4+ T cells, Tregs, endothelial cells, neutrophils, and M2-type macrophage quantification (MCQ) immune infiltration. However, KLF8 sowed significant negative correlations with the infiltration of CD8+ T cells, NK cells, and myeloid-derived suppressor cells (MDSC) (Figure 8B).\n\n(A) KLF3 expression and immune infiltrating cells abundance of CD4+ T cells, CD4+ T cells, neutrophils, Tregs, M2 MCQ and NK cells.\n\n(B) KLF8 expression and immune infiltrating cells abundance of CD4+ T cells, CD4+ T cells, neutrophils, Tregs, M2 MCQ, NK cells, endothelial cells and myeloid-derived suppressor cells (MDSC).\n\nThese findings provide insights into the relationships between autophagy genes, KLF3, KLF8, and various immune cell populations in the tumor microenvironment of CRC. The correlations observed highlight the potential roles of these genes in immune cell infiltration and the modulation of the tumor immune response.\n\n\nDiscussion\n\nCRC invasion and metastasis are major contributors to high mortality rates and tumor recurrence.45 Therefore, understanding the underlying processes involved in these malignant phenotypes is crucial to developing therapeutic targets and overcoming chemotherapy resistance.\n\nMost CRC cell line were found to have a strong baseline autophagic flux. However, previous literature showed that HCT 116 showed a higher controlled autophagic flux compared to other CRC cell lines.40 Thus, we implemented our CRISPR/Cas9 experiments on HCT 116 cell lines specifically to study the effect of autophagy inhibition efficiently. Moreover, targeting ATG5/7, which are specific and crucial autophagy genes, is essential to block the conventional autophagy pathway. Specifically, in HCT 116, was associated with a dramatic decrease in cell survival when targeting ATG5 or ATG7.46 All gene editing techniques are predicted to have an off-target effect. From our side, we used the ribonucleoprotein-CRISPR Cas9 method, which has been proven to have a lower off-target effect compared to the plasmid delivery system since it limits the activity time for Cas9.47 The sgRNAs used were selected based on the top scores or high scores according to the Thermoscientific Fischer system of sgRNA designing. Using this system allowed us to produce a few knockout (KO) clones; however, one clone was taken further for downstream experimentation.\n\nThe relationship between autophagy and its role and interaction with KLF3 and KLF8 in CRC remains poorly characterized.48 Furthermore, the correlation between the KLF family, specifically KLF3 and KLF8, and autophagy has not been previously studied in tumors, presenting an opportunity for novel therapeutic approaches in CRC. In this study, we aimed to investigate the expression levels of multiple autophagy genes in relation to KLF3 and KLF8 in CRC, as well as their clinicopathological features, utilizing various web tools and large RNA sequence datasets obtained from cancer patients.\n\nOur experimental investigation found that KLF8 and KLF3 expression was significantly higher in the late stages of CRC, indicating its strong association with enhanced EMT and metastasis. Specifically, the mRNA expression level of KLF8 was significantly higher in metastatic cell lines such as LoVo and SK-CO-1 compared to HCT 116 parental cell line, however, KLF3 expression was significantly higher in LoVo cell line. KLF8 has been found to play a role in modulating metabolism in gastric cancer by targeting GLUT4, thereby increasing glucose uptake crucial for cancer cell proliferation and metastasis.49 Likewise, KLF3 has been recognized as an oncogenic transcription factor in CRC with a significant impact on the regulation of tumorigenesis.50\n\nBased on our transcriptomic analysis and the findings from our bioinformatics analysis, we have identified a strong correlation between the transcriptional factors KLF3 and KLF8 and the expression of multiple autophagy genes in CRC. This correlation has been addressed in cancer cells for the first time, providing valuable insights into the potential crosstalk between KLF3/KLF8 and autophagy in CRC. Interestingly, our investigation revealed that the inhibition of autophagy in ATG5-/- and ATG7-/- cell lines resulted in differential expression of KLF3, suggesting that it may act as a compensatory mechanism in response to autophagy suppression. It is worth noting that KLF3 and KLF8 are highly related transcriptional regulators that bind to similar DNA sequences and exhibit overlapping roles.51 Because autophagy is required for KLF3 degradation, its expression was elevated in our transcriptomic analysis in the KO cell lines. The 3-MA treatment was found to impede the turnover of KLF2 and KLF3.26 Moreover, according to PubChem database,; the two proteins have a high homology.52 Notably, KLF8 transcriptional repression activity is triggered by its interaction with CtBP.53 KLF8 performs transcriptional activation in addition to transcriptional repression. KLF8’s glutamine residues Q118 and Q248 are crucial for the protein’s transcriptional activation function.54\n\nAutophagy has been implicated in the invasion and metastasis processes, prompting us to investigate the association between autophagy genes and different epithelial-mesenchymal transition (EMT) markers, which play a crucial role in the metastasis of colorectal cancer (CRC). Our analysis revealed a strong correlation between autophagy genes and various EMT markers, underscoring their interconnectedness in CRC progression. Furthermore, both KLF3 and KLF8 showed significant positive correlations with multiple EMT markers. Notably, KLF8 exhibited the highest association with the ZEB1 gene (p = 3.1e-52). This finding is particularly interesting, considering that UVRAG, an autophagy gene, displayed the strongest positive correlation with the critical EMT marker ZEB1. Remarkably, KLF8 is associated with decreased E-cadherin. Overexpression of KLF8 decreases E-cadherin expression, promoting invasion and serving as a possible EMT inducer. In non-small cell lung cancer, decreased KLF8 expression prevents malignancy through preventing cell invasion and EMT.55 Previous studies have shown that KLF8 induces FHL2-mediated invasion, EMT, and metastasis in CRC.56 In gastric cancer, KLF8 has been implicated in promoting invasion and metastasis via the EMT process by targeting the hypoxia-induced factor (HIF-1) gene.57 The observed correlations provide insights into the potential mechanisms underlying the role of autophagy and KLF8 in the invasion, metastasis, and EMT, offering opportunities for further exploration and potential therapeutic interventions targeting these pathways.\n\nConsistent with our findings, previous studies have demonstrated the necessity of autophagy for EMT activation and metastasis in various cancer types. For example, autophagy is required for EMT activation and metastasis in hepatoblastoma cells58 as well as in TGF-β1-mediated EMT in non-small-cell lung carcinoma cells.59 Thus, despite the contextual relationship between the complex autophagy machinery and cancer, it is still a promising target, as there are numerous common regulatory pathways between autophagy and cancer. Tumor samples with high ULK2 and UVRAG were found to be significantly associated with mucinous colorectal adenocarcinoma, indicating unfavorable prognosis.\n\nTME plays a crucial role in modulating the rate of invasion and metastasis in colorectal cancer (CRC). The interaction between CRC cells and their microenvironment is influenced by various factors, with the tumor microsatellite status (MSI vs. MSS) being one of the key determinants. Patients with MSS tumors generally have a worse prognosis compared to those with MSI tumors.60 Using the GENT2 web tool,34 our study observed that KLF8 expression was significantly higher in MSS tumors, while KLF3 expression was higher in MSI tumors. This finding was further validated through qPCR analysis, where we confirmed that the MLH1+/- cell line, representing the MSS subtype, exhibited significantly higher KLF8 expression than the HCT 116 cell line, representing the MSI phenotype. Importantly, KLF8 has been identified as a novel regulator of DNA damage response pathways in breast cancer.61 Ineffective DNA repair can lead to genomic instability, which impacts cellular functions and contributes to cancer development. KLF8 interacts with PARP-1 and other DNA damage regulators, forming a complex that influences cellular functions and alters DNA repair, thereby affecting genomic stability and contributing to enhanced cancer progression.61,62 However, further investigations are warranted to elucidate the specific role of KLF8 in the context of MSS phenotype in CRC. Understanding the involvement of KLF8 in regulating DNA repair pathways and its impact on genomic instability could provide valuable insights into the mechanisms underlying MSS CRC progression and potential therapeutic strategies.\n\nThe MSI and MSS subtypes have distinct immunogenic microenvironments besides variable genomic instability. It has been established that the MSI tumors had more immune infiltrating cells than the MSS tumors, which typically have a higher abundance of immune infiltrating cells than MSS tumors.63 Among these immune cells, cytotoxic CD8+ T-lymphocytes (CTL) are considered the primary defense mechanism against tumor cells. The abundance of T cells is a significant and important prognostic indicator for the effectiveness of immunotherapy and chemotherapy.64 Regulatory T-cells (Tregs) are another crucial form of T-cells closely associated with colorectal tumors.\n\nHigh infiltration of immune cells, including CD8+, CD4+, neutrophils and NK cells, was correlated with KLF3 expression in our analysis. In contrast, KLF8 expression was significantly negatively correlated with CD8+ and NK cells, whereas it was significantly positively correlated with Tregs and M2-type macrophages. These results suggest that KLF3 and KLF8 can potentially serve as prognostic indicators for immunotherapy efficacy. The differential associations of these transcription factors with specific immune cell populations highlight their potential role in shaping the tumor microenvironment and influencing the response to immunotherapy in CRC.\n\nAutophagy has a major influence on the tumor-specific CD8+ T cells65 and is found to be crucial for the activity of effector and memory T-cells.66 However, the role of autophagy in tumor immunity is complex and can have both promoting and inhibitory effects on tumor progression. Autophagy has been shown to contribute to the degradation of cytolytic granules secreted by cytotoxic CD8+ T cells and natural killer (NK) cells, thereby dampening their antitumor activity.67,68 Additionally, autophagy plays a role in the immunosuppressive function of regulatory T (Treg) cells, which rely on autophagy for their suppressive effect.69 In the tumor microenvironment, tumor-associated macrophages (TAMs) of the M2 phenotype have been shown to promote tumor growth, angiogenesis, and metastasis.70\n\nConversely, M1 macrophages have been implicated in inhibiting tumor progression.71 Autophagy has been found to participate in the production and polarization of macrophages, with Toll-like receptor-2 (TLR2) deficiency resulting in impaired autophagy and the generation of M2-type macrophages that support tumor progression.72 Furthermore, autophagy induction in TAMs has been shown to promote apoptotic cell death, restrain proliferation, and enhance radiosensitivity in colorectal cancer.73 These findings highlight the complex role of autophagy in modulating the tumor microenvironment and its impact on cancer progression. It is important to note that the role of autophagy in cancer is dynamic and can vary depending on the specific context and stage of tumor development. While our bioinformatics analysis using the TIMER web tool explored the correlation between immune infiltrating cells and autophagy genes in a comprehensive CRC patient dataset, further studies are needed to investigate the specific subtypes and stages of CRC to gain a deeper understanding of the relationship between autophagy and the immune microenvironment in different contexts.\n\nIn colorectal cancer (CRC), the differential expression of autophagy genes is likely regulated through epigenetic mechanisms rather than gene mutations. In the presence of microsatellite instability (MSI), alterations in the crucial autophagy gene UVRAG can lead to truncated mutations, resulting in dysregulated UVRAG expression and subsequent tumor growth.74 Similarly, KLF8 has been found to have an amplification mutation and higher mRNA levels in CRC compared to KLF3. To further investigate the regulation of gene expression, we evaluated the promoter methylation levels of KLF3 and KLF8. Promoter methylation is known to affect mRNA expression by influencing the accessibility of DNA to transcriptional machinery. Previous studies in prostate cancer cells have shown a negative association between DNA methylation at the promoter region of various KLF genes, including KLF3 and KLF8, and their mRNA expression levels.75 In our analysis, we observed a significant decrease in KLF3 promoter methylation levels in late CRC stages. In contrast, KLF8 promoter methylation levels were significantly decreased in stage 4 CRC and late stages of lymph node metastasis.\n\nAutophagy, in this context, promotes cell survival and progression by suppressing cell death.28,76 In various cancers, including colorectal cancer (CRC), a significant correlation has been observed between the expression of autophagy genes and the activation of the RAS-MAPK pathway. Specifically, ATG7 has been found to correlate with the activation of the RAS-MAPK pathway in CRC. Furthermore, autophagy has been implicated in activating epithelial-to-mesenchymal transition (EMT), a critical process in tumor progression and metastasis. While many autophagy genes are generally correlated with EMT activation in different cancer types, specific to CRC, UVRAG and ULK2 have shown a significant correlation with EMT activation. These findings highlight the intricate relationship between autophagy and key signaling pathways, such as RAS-MAPK and EMT, in promoting cancer development and progression. Targeting autophagy, particularly in the context of these specific pathways, may hold promise for future therapeutic interventions in CRC and other cancers.\n\nOne of the most potent inducers of EMT is transforming growth factor-beta (TGF-β), which activates both Smad and non-Smad signaling pathways. Notably, KLF8 has been identified as one of the downstream targets of TGF-β signaling.77 Inhibition of extracellular signal-regulated kinase (ERK) signaling has been shown to reduce both KLF8 expression and cellular motility.78\n\nThe primary limitation of our study lies in its reliance on a single cell line, HCT 116 to perform CRISPR/Cas9 gene editing technique to knockout ATG5/7. However, we used several cell lines in our experiments. Generalizing our findings to broader biological scenarios should be approached with caution, recognizing the need for further investigation across multiple cell lines to enhance the robustness and applicability of our results.\n\n\nConclusions\n\nThe current study highlights the potential therapeutic significance of targeting both KLF8 and autophagy in treating metastatic colorectal cancer (CRC), in particular, MMR deficient subtypes. A synergistic effect may be achieved by simultaneously inhibiting KLF8, which is implicated in cellular motility, proliferation, and transcriptional regulation, and autophagy, which has been linked to tumor progression and resistance to chemotherapy. This combined approach could potentially enhance treatment efficacy by targeting multiple pathways involved in CRC metastasis and chemoresistance. Further studies are warranted to explore the feasibility and effectiveness of this therapeutic strategy in preclinical and clinical settings.\n\n\nEthical approval and consent\n\nEthical approval and consent were not required.\n\n\nAuthors contribution\n\nEglal Mahgoub: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jalal Taneera: visualization and revision. Samrein Ahmed: for CRISPR-Cas9 gene editing experiments and data analysis. Shirin Hafezi: Experimental work (CRISPR-Cas9) and data analysis. Thenmozhi Venkatachalam: Transcriptomics analysis, Methodology, writing. Mahmood Hachim: Bioinformatics analysis. Nabil Sulaiman: Reviewing. Rifat Hamoudi: Bioinformatics Analysis and revision. Maha Saber-Ayad: Conceptualization, Writing – review & editing, Supervision, Resources, Methodology, Project administration, Funding acquisition.", "appendix": "Data availability\n\nFigshare: Unraveling the interplay of autophagy genes and KLF3/KLF8 in Colorectal cancer metastasis: A bioinformatics and cellular exploration, https://doi.org/10.6084/m9.figshare.25655457.v2. 41\n\nThis project contains the following underlying data:\n\n• Data file: The data is whole transcriptomics analysis (RNAseq) of 7 samples of isogenic colorectal cancer cell line HCT116 (parental wild HCT116, HCT116_negatic control, HCT116_Knocked out for ATG5, HCT116_Knocked out for ATG7). The cells were knocked out using Caspase Cas9 (ribonucleoprotein). RNA-seq data were analyzed using the Ion Torrent Software Suite version and the alignment was carried out using the Torrent Mapping Alignment Program (TMAP).\n\n• Data file: Full western blot gel for protein expression of ATG5 and ATG7 in HCT 116, HT29, LoVo and SK-CO-1).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgments\n\nWe would like to acknowledge the support of the University of Sharjah.\n\n\nReferences\n\nBray F, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018; 68(6): 394–424. PubMed Abstract \| Publisher Full Text\n\nDouaiher J, et al.: Colorectal cancer—global burden, trends, and geographical variations. J. Surg. Oncol. 2017; 115(5): 619–630. PubMed Abstract \| Publisher Full Text\n\nYang A, et al.: Autophagy Is Critical for Pancreatic Tumor Growth and Progression in Tumors with p53 AlterationsAutophagy Is Critical for Pancreatic Tumor Growth. Cancer Discov. 2014; 4(8): 905–913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalluzzi L, et al.: Molecular definitions of autophagy and related processes. EMBO J. 2017; 36(13): 1811–1836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMizushima N: Autophagy: process and function. Genes Dev. 2007; 21(22): 2861–2873. Publisher Full Text\n\nMizushima N, Klionsky DJ: Protein turnover via autophagy: implications for metabolism. Annu. Rev. Nutr. 2007; 27(1): 19–40. Publisher Full Text\n\nWhite E, DiPaola RS: The double-edged sword of autophagy modulation in cancer. Clin. Cancer Res. 2009; 15(17): 5308–5316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoya P, Reggiori F, Codogno P: Emerging regulation and functions of autophagy. Nat. Cell Biol. 2013; 15(7): 713–720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite E: Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer. 2012; 12(6): 401–410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDegenhardt K, et al.: Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006; 10(1): 51–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolyak K, Weinberg RA: Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat. Rev. Cancer. 2009; 9(4): 265–273. Publisher Full Text\n\nDi Malta C, Cinque L, Settembre C: Transcriptional regulation of autophagy: mechanisms and diseases. Front. Cell Dev. Biol. 2019; 7: 114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi X, He S, Ma B: Autophagy and autophagy-related proteins in cancer. Mol. Cancer. 2020; 19(1): 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTetreault M-P, Yang Y, Katz JP: Krüppel-like factors in cancer. Nat. Rev. Cancer. 2013; 13(10): 701–713. Publisher Full Text\n\nMcConnell BB, Yang VW: Mammalian Krüppel-like factors in health and diseases. Physiol. Rev. 2010; 90(4): 1337–1381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsieh PN, et al.: A conserved KLF-autophagy pathway modulates nematode lifespan and mammalian age-associated vascular dysfunction. Nat. Commun. 2017; 8(1): 914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa D, et al.: KLF8 overexpression promotes the growth of human lung cancer cells by promoting the expression of JMJD2A. Cancer Cell Int. 2019; 19(1): 1–10. Publisher Full Text\n\nLiang K, et al.: KLF8 is required for bladder cancer cell proliferation and migration. Biotechnol. Appl. Biochem. 2015; 62(5): 628–633. PubMed Abstract \| Publisher Full Text\n\nWang W-F, et al.: Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer. World J. Gastroenterol. 2013; 19(27): 4309–4315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYi X, et al.: KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells. Gene. 2016; 585(1): 22–27. PubMed Abstract \| Publisher Full Text\n\nLiu J, et al.: The level of Krüppel-like factor 8 expression predicts prognosis and metastasis in various carcinomas. Medicine. 2019; 98(18): e15519. Publisher Full Text\n\nWang X, et al.: KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene. 2011; 30(16): 1901–1911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVliet J, Turner J, Crossley M: Human Kruppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Res. 2000; 28(9): 1955–1962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, et al.: Protein database searches using compositionally adjusted substitution matrices. FEBS J. 2005; 272(20): 5101–5109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo L, et al.: Transactivation of Atg4b by C/EBPβ promotes autophagy to facilitate adipogenesis. Mol. Cell. Biol. 2013; 33(16): 3180–3190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomlinson JS, et al.: Actual 10-year survival after resection of colorectal liver metastases defines cure. J. Clin. Oncol. 2007; 25(29): 4575–4580. PubMed Abstract \| Publisher Full Text\n\nKinsey CG, et al.: Protective autophagy elicited by RAF→ MEK→ ERK inhibition suggests a treatment strategy for RAS-driven cancers. Nat. Med. 2019; 25(4): 620–627. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTalaat IM, et al.: Evaluation of Galanin Expression in Colorectal Cancer: An Immunohistochemical and Transcriptomic Study. Front. Oncol. 2022; 12: 12. Publisher Full Text\n\nNing Z, Cox AJ, Mullikin JC: SSAHA: a fast search method for large DNA databases. Genome Res. 2001; 11(10): 1725–1729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly. Bioinformatics. 2012; 28(14): 1838–1844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith TF, Waterman MS: Identification of common molecular subsequences. J. Mol. Biol. 1981; 147(1): 195–197. PubMed Abstract \| Publisher Full Text\n\nLi T, et al.: TIMER2. 0 for analysis of tumor-infiltrating immune cells. Nucleic Acids Res. 2020; 48(W1): W509–W514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaham AJ, et al.: A bioinformatics evaluation of the role of dual-specificity tyrosine-regulated kinases in colorectal cancer. Cancers. 2022; 14(8): 2034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark S-J, et al.: GENT2: an updated gene expression database for normal and tumor tissues. BMC Med. Genet. 2019; 12(5): 1–8. Publisher Full Text\n\nTang Z, et al.: GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res. 2019; 47(W1): W556–W560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChandrashekar DS, et al.: UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia. 2017; 19(8): 649–658. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C-J, et al.: GSCALite: a web server for gene set cancer analysis. Bioinformatics. 2018; 34(21): 3771–3772. PubMed Abstract \| Publisher Full Text\n\nFranz M, et al.: GeneMANIA update 2018. Nucleic Acids Res. 2018; 46(W1): W60–W64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLauzier A, et al.: Colorectal cancer cells respond differentially to autophagy inhibition in vivo. Sci. Rep. 2019; 9(1): 11316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamoudi R, Mahgoub E, Taneera J, et al.: Unraveling the interplay of autophagy genes and KLF3/KLF8 in Colorectal cancer metastasis: A bioinformatics and cellular exploration. Dataset. figshare. 2024. Publisher Full Text\n\nMüller JA, et al.: SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas. Nat. Metab. 2021; 3(2): 149–165. PubMed Abstract \| Publisher Full Text\n\nRojas-Sanchez G, et al.: Autophagy and Its Relationship to Epithelial to Mesenchymal Transition: When Autophagy Inhibition for Cancer Therapy Turns Counterproductive. Biology. 2019; 8(4): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBustos SO, et al.: Emerging autophagy functions shape the tumor microenvironment and play a role in cancer progression-implications for cancer therapy. Front. Oncol. 2020; 10: 606436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlSafar H, et al.: COVID-19 disease severity and death in relation to vitamin D status among SARS-CoV-2-positive UAE residents. Nutrients. 2021; 13(5): 1714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen L, et al.: Mechanisms on chemotherapy resistance of colorectal cancer stem cells and research progress of reverse transformation: A mini-review. Front. Med. 2022; 9. Publisher Full Text\n\nGuo JY, et al.: Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 2011; 25(5): 460–470. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang S, et al.: Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics. 2021; 11(2): 614–648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenific CM, Thorburn A, Debnath J: Autophagy and metastasis: another double-edged sword. Curr. Opin. Cell Biol. 2010; 22(2): 241–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMao A, et al.: KLF8 is associated with poor prognosis and regulates glycolysis by targeting GLUT4 in gastric cancer. J. Cell. Mol. Med. 2019; 23(8): 5087–5097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q-L, et al.: Genome-wide profiling in colorectal cancer identifies PHF19 and TBC1D16 as oncogenic super enhancers. Nat. Commun. 2021; 12(1): 6407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFunnell AP, et al.: Generation of mice deficient in both KLF3/BKLF and KLF8 reveals a genetic interaction and a role for these factors in embryonic globin gene silencing. Mol. Cell. Biol. 2013; 33(15): 2976–2987. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, et al.: PubChem 2019 update: improved access to chemical data. Nucleic Acids Res. 2019; 47(D1): D1102–D1109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao J, et al.: Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol. Cell. 2003; 11(6): 1503–1515. PubMed Abstract \| Publisher Full Text\n\nUrvalek AM, et al.: KLF8 recruits the p300 and PCAF co-activators to its amino terminal activation domain to activate transcription. Cell Cycle. 2010; 9(3): 601–611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi H, et al.: MiR-135a inhibits migration and invasion and regulates EMT-related marker genes by targeting KLF8 in lung cancer cells. Biochem. Biophys. Res. Commun. 2015; 465(1): 125–130. PubMed Abstract \| Publisher Full Text\n\nYan Q, et al.: KLF8 promotes tumorigenesis, invasion and metastasis of colorectal cancer cells by transcriptional activation of FHL2. Oncotarget. 2015; 6(28): 25402–25417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu N, et al.: Krüppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of gastric cancer via epithelial to mesenchymal transition. Oncol. Rep. 2014; 32(6): 2397–2404. PubMed Abstract \| Publisher Full Text\n\nChen C, et al.: DRAM1 regulates the migration and invasion of hepatoblastoma cells via autophagy-EMT pathway. Oncol. Lett. 2018; 16(2): 2427–2433. PubMed Abstract \| Publisher Full Text\n\nAlizadeh J, et al.: Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells. Biochimica et Biophysica Acta (BBA)-Molecular. Cell Res. 2018; 1865(5): 749–768. Publisher Full Text\n\nKawakami H, Zaanan A, Sinicrope FA: Microsatellite instability testing and its role in the management of colorectal cancer. Curr. Treat. Options Oncol. 2015; 16: 1–15.\n\nLu H, et al.: A novel role of Krüppel-like factor 8 in DNA repair in breast cancer cells. J. Biol. Chem. 2012; 287(52): 43720–43729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu H, et al.: Identification of poly (ADP-ribose) polymerase-1 (PARP-1) as a novel Krüppel-like factor 8-interacting and-regulating protein. J. Biol. Chem. 2011; 286(23): 20335–20344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiao Y, Freeman GJ: The microsatellite instable subset of colorectal cancer is a particularly good candidate for checkpoint blockade immunotherapy. Cancer Discov. 2015; 5(1): 16–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBupathi M, Wu C: Biomarkers for immune therapy in colorectal cancer: mismatch-repair deficiency and others. J. Gastrointest. Oncol. 2016; 7(5): 713–720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUhl M, et al.: Autophagy within the antigen donor cell facilitates efficient antigen cross-priming of virus-specific CD8+ T cells. Cell Death Differ. 2009; 16(7): 991–1005. PubMed Abstract \| Publisher Full Text\n\nXu X, et al.: Autophagy is essential for effector CD8+ T cell survival and memory formation. Nat. Immunol. 2014; 15(12): 1152–1161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaginska J, et al.: Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia. Proc. Natl. Acad. Sci. 2013; 110(43): 17450–17455. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoman MZ, et al.: Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression. Cancer Res. 2011; 71(18): 5976–5986. PubMed Abstract \| Publisher Full Text\n\nWei J, et al.: Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nat. Immunol. 2016; 17(3): 277–285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaldiero MR, et al.: Tumor associated macrophages and neutrophils in cancer. Immunobiology. 2013; 218(11): 1402–1410. Publisher Full Text\n\nMizuno R, et al.: The role of tumor-associated neutrophils in colorectal cancer. Int. J. Mol. Sci. 2019; 20(3): 529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang M, et al.: Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization. Mol. Cancer. 2014; 13(1): 1–15.\n\nShao LN, et al.: Effects of autophagy regulation of tumor-associated macrophages on radiosensitivity of colorectal cancer cells. Mol. Med. Rep. 2016; 13(3): 2661–2670. PubMed Abstract \| Publisher Full Text\n\nDrucker A, et al.: Ephrin b2 receptor and microsatellite status in lymph node-positive colon cancer survival. Transl. Oncol. 2013; 6(5): 520–527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeng J, et al.: Characterization of the prognostic values and response to immunotherapy/chemotherapy of Krüppel-like factors in prostate cancer. J. Cell. Mol. Med. 2020; 24(10): 5797–5810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee C-S, et al.: MAP kinase and autophagy pathways cooperate to maintain RAS mutant cancer cell survival. Proc. Natl. Acad. Sci. 2019; 116(10): 4508–4517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrelet S, et al.: Pleiotropic roles of non-coding RNAs in TGF-β-mediated epithelial-mesenchymal transition and their functions in tumor progression. Cancers. 2017; 9(7): 75. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "309841", "date": "13 Sep 2024", "name": "Aamir Ahmed", "expertise": [ "Reviewer Expertise Cell signalling", "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\nUnlike the unexceptional sequencing manuscripts that abound, currently, this is an interesting investigation that uses gene knockout followed by transcriptomic analysis of HCT 116 knockout and correlates certain significant cellular pathways in colorectal carcinogenesis. The study is largely exploratory without an overarching hypothesis, however, it is well designed and provides useful new information to justify publication. I have the following suggestions for the authors to consider in their revision. There are two aspects to this (1) notions that could be incorporated into a revised manuscript mostly by further analysis of the transcriptomic data that they have already accumulated (2) to consider adding simple biochemical aspects to provide a true biological validation of their conclusions. 1. Additional analysis of sequencing dataset: 1.1. The authors show an interest in autophagy in CRC. Autophagy is a well investigated mechanism in eukaryotes from yeast to man. There are three main types of autophagy in mammalian cells with distinct mechanistic operations, namely, micro-, macro- and chaperone mediated autophagy. The authors may wish to interrogate and classify these in their knockout cells to identify if these are differentially manifested. It would then be important to discover if different grades of cancer show any difference associated with at least the three key types of autophagy. 1.2. Autophagy has been extensively studied in model organisms such as yeast (e.g. Reggiori, F et al2013,)[ Ref 1]. It would give this manuscript another and useful dimension if the authors compare genetic regulation of autophagy in yeast compared to their dataset (https://www.yeastgenome.org/search?q=autophagy&category=dataset).\n2. Biochemical validation of knockout cells with regards to autophagy: The abstract states that “.....which revealed an upregulation of Krϋppel-like factor 3 (KLF3) expression, that was biologically validated”.\nI am not sure what is exactly meant by ‘biological validation’? The only reference I found in the manuscript related to qPCR confirmation data. The authors have an excellent opportunity to complement their sequencing analysis with a true biological validation of the mechanism of autophagy and how it may relate to colorectal cancer cell lines. As a paradigm for biological validation the authors should consider the following experiments.\n2.1. mTOR kinase: I was surprised to see that there is no discussion of this kinase in the manuscript which is a key mechanistic link for autophagy in cells. For example, it is known that over-expression of members of KLF family inhibits mTOR (mammalian Target Of Rapamycin) activity (e.g. Wang, Y. et al 2012) [Ref 2]. It is important that an assessment of mTOR activity is made in the knockout cell lines. A rather simple way of doing this would be, for example, to measure the phosphorylation status of ribosomal S6 protein.\n2.2. Autophagy and mTOR are in turn tightly linked to the nutritional status of cells. It will be important to know if the ATG knockout cells exhibit alterations in growth patterns or the nutritional status?\n2.3. Most genetic regulation of carcinogenesis involves mutation or disruption, not complete knockout of genes, which is rare. This presents a problem for the extrapolation of gene knockout data from model systems (e.g. cell lines) to the human disease as it is the gene dose which could be critical in the evolution of disease. An important biological validation of the authors’ gene knockout model would be to conduct experiments where the protein products of two autophagy genes(ATG5 and ATG7) are inhibited in the cell lines followed bymtranscriptomics. A comparison of the transcriptome from knockout vs inhibition model strengthen the manuscript. Furthermore, it should be noted that for enzymes such as ligases and kinases, it is not the gene expression levels that are critical, rather it is the activity of these enzymes that determine the functional outcome in cells. The authors need to address these issues in their revision.\nOther comments: I noticed some errors in the text of the manuscript. A few examples are highlighted below, however, I suggest that the authors conduct a thorough edit of the manuscript. 1. “However, KLF8 sowed significant negative correlations with the infiltration of CD8+ T cells, NK cells” Comment: Spelling\n2. Regulation of autophagy is mediated via several transcription factors, or a transcriptional gene network. Add that autophagy inhibition did not show a marked suppressing effect on cancer cell proliferation, however, compelling evidence supports the notion that autophagy actively contributes to the migration and invasion of cancer cells. Comment: Non-sequitur, grammar\n3. “Autophagy, a cellular process, plays a significant role in the development of CRC and renders cancer cells less susceptible to chemotherapy”. “Autophagy may act as a tumor suppressor in the early stages of tumor formation, but it appears to promote tumor growth once tumors have already developed”. Comment: Citation for these statements please\n4. “KLF3/KLF8 expression level was elevated in late stages of CRC cells” Comment: You mean CRC as in cancer not CRC cell lines?\n- I have not come across any supplementary data. Although not always adhered to but the original sequencing dataset should be provided.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "332099", "date": "05 Nov 2024", "name": "Mohammad Azhar Aziz", "expertise": [ "Reviewer Expertise Colorectal cancer 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\nThis manuscript aims to establish relationship among the autophagy genes and transcriptions factors KLF3/8 in a metastatic colorectal cancer model. The manuscript undertakes several computational approaches to delineate the relationship and used their own cell lines with ATG5/7 knockout generated using CRISPR/Cas9. This study needs a complete redesign and following points would be helpful. However, these are just examples to prompt the authors about redesigning their study for proper conclusions, there are many other instances which would need attention. The figures are of poor quality and hence does not allow proper understanding of data. Figures are not even readable. Some of my comments may be due to poor figure qualities (Figures 1-4). 1. There is an existing pair of cell line SW480/620 which would serve as a better model for studying CRC progression into metastasis. Comparing HCT116 with LOVO and SK-CO-1 may not be the right testing model. While LOVO is stage 4 representation, what is the purpose for SK-CO-1?. Please provide reference or evidence that SK-CO-1represents metastatic stage. On ATCC website its mentioned that it was derived from Colorectal Adenocarcinoma? 2. Was it ascertained that there is no change of GAPDH before using it as housekeeping control for qRT-PCR?? This can be done by using the transcriptomics data. 3. Figure 1 does not show any marking with the arrows. Its very low resolution that makes it impossible to understand any result given in Figure legend. Why there is no discussion about the TBC1D22B gene along with KLF3?? 4. One copy of MLH1 knock in is insufficient evidence for MSS stability. Although it has been used earlier but not suitable for gene expression studies where gene copy number is numerically linked to mRNA levels. 5. Figure 2: KLF3 comparison is between Stage 1 and 2/3 whereas for KLF8 its only 1 and 4. Why? Why methylation patterns do not correlate with expression level. Figure 2 does not conclude that KLF3/8 are associated with 'progression' of CRC. It just shows expression of KLF3/8 at a particular stage of CRC. Progression evidence would need the study that shows halting of progression through abolishing the KLF3/8 function. 6. Figure 3: What is the KLF3/8 levels in ATG5/7 knockout cell line? Where is the data for transcriptomic analysis of the KO cell lines?? 7. Figure 4: Where is the data for EMT markers in ATG5/7 knockout cell line?? 8. KLF3 is known to effect the Wnt Signaling pathway which is crucial for CRC. However, it has not been analyzed or discussed anywhere. Why?? Authors should redesign the study and provide complete data to allow independent analysis before concluding the inhibition of KLF3 and autophagy genes as therapeutic targets.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/13-850
https://f1000research.com/articles/13-481/v1	17 May 24	{ "type": "Data Note", "title": "A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence", "authors": [ "Riham Ayoubi", "Maryam Fotouhi", "Charles Alende", "Sara González Bolívar", "Kathleen Southern", "Carl Laflamme", "Neuro/SGC/EDDU collaborative group", "ABIF consortium", "Riham Ayoubi", "Maryam Fotouhi", "Charles Alende", "Sara González Bolívar", "Kathleen Southern" ], "abstract": "Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.", "keywords": [ "Uniprot ID P21980", "TGM2", "TGM", "Protein-glutamine gamma-glutamyltransferase 2", "antibody characterization", "antibody validation", "western blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nProtein-glutamine gamma glutamyltransferase 2 (TGM2) belongs to the transglutaminase family of Ca2+ dependent enzymes, regulating various cellular processes, including cell differentiation, growth and apoptosis.1–3 Encoded by TGM2 gene, TGM2 protein exhibits its function through Ca2+-dependent cross-linking of substrates, thereby modulating their activity.1 TGM2’s catalytic activity is dependent on guanine nucleotides and Ca2+ binding.4 Both GTP and/or GDP act as negative regulators of TGM2, inducing a conformational change upon binding, inhibiting its cross-linking activity (closed form). Conversely, Ca2+ binding prompts a conformational change to induce TGM2 activity (open form). The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion.5–9\n\nAlteration and regulation of TGM2’s activity is associated with the pathogenesis of various diseases including cancer, neurodegeneration, fibrosis, inflammatory, autoimmune disorders and liver diseases.10–13 Increased TGM2 mRNA transcripts, resulting in elevated transaminase enzymatic activity, have been associated with neurodegenerative mechanism observed in Parkinson’s disease, Alzheimer’s disease and Huntington’s disease.14,15 Given that α-synuclein serves as a common substrate for TGM2, elevated activity of TGM2 results in the formation of soluble aggregates as well as insoluble inclusions; distinctive features of Parkinson’s disease pathogenesis.15–18 Regulating TGM2 activity using inhibitors could positively affect the human diseases in which TGM2 is implicated.19,20 Identifying high-quality antibodies would accelerate TGM2 research and its potential as a pharmacological target.\n\nThis research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data.21–23 Here, we evaluated the performance of seventeen commercially-available antibodies for TGM2 for use in western blot, and sixteen for immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TGM2 properties and function. The platform for antibody characterization used to carry out this study was approved by a committee of industry, academic research. It consists of first identifying appropriate human cell lines, development/contribution of equivalent knockout cell lines and finally following antibody characterization procedures on commonly used commercial antibodies. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange, DOI: 10.21203/rs.3.pex-2607/v1.24\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.25,26 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available A549 cells expressed the TGM2 transcript at RNA levels above the average range of cancer cells analyzed. Parental and TGM2 KO A549 cells were obtained from Abcam (Table 1).\n\nFor western blot experiments, we resolved proteins from WT and TGM2 KO cell extracts and probed them side-by-side with all antibodies in parallel on lysate and culture medium (Figures 1 and 2).\n\nLysates of A549 (WT and TGM2 KO) were prepared and 40 μg of protein were processed for western blot with the indicated TGM2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab109121 at 1/1000, ab109200 at 1/10000, ab2386* at 1/500, ab310333 at 1/1000, ab421 at 1/500, ABCD_AI748 at 1/10, A21184** at 1/10000, ARP47471 at 1/500, ARP47472 at 1/500, AF4376 at 1/400, MAB4376* at 1/200, 3557** at 1/500, GTX111702 at 1/500, 15100-1-AP at 1/6000, 68006-1-Ig* at 1/10000, MA5-32819** at 1/500, MA5-12739* at 1/200. Predicted band size: 77 kDa. Monoclonal antibody, Recombinant antibody.\n\nA549 WT and TGM2 KO were cultured in serum free media, and 40 μg of protein from concentrated culture media were processed for western blot with the indicated TGM2 antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilution used: ab109121* at 1/1000, ab109200** at 1/10000, ab2386* at 1/500, ab310333 at 1/1000, ab421 at 1/500, ABCD_AI748 at 1/10, A21184** at 1/10000, ARP47471 at 1/500, ARP47472 at 1/500, AF4376 at 1/400, MAB4376* at 1/200, 3557** at 1/500, GTX111702 at 1/500, 15100-1-AP at 1/6000, 68006-1-Ig* at 1/10000, MA5-32819** at 1/500, MA5-12739* at 1/200. Predicted band size: 77 kDa. Monoclonal antibody, Recombinant antibody.\n\nAs per our standard procedure, we next used the antibodies to immunoprecipitate TGM2 from A549 cell extracts. To evaluate the performance of each antibody, the TGM2 protein was detected in extracts, in each extract unbound to the antibody and corresponding immunoprecipitate (Figure 3). To detect TGM2, a western blot was performed with an antibody successful under the conditions tested in Figure 1.\n\nA549 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated TGM2 antibodies pre-coupled to Dynabeads protein A or protein. Samples were washed and processed for western blot with the indicated TGM2 antibody. For western blot, A21184* was used at 1/10000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; n/s=non-specific signal. Monoclonal antibody, Recombinant antibody.\n\nFor immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure. First, A549 WT and TGM2 KO were labelled with different fluorescent dyes in order to distinguish the two cell lines, and TGM2 antibodies were evaluated. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 4). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested. The images presented in Figure 4 are representative of the results of this analysis.\n\nA549 WT and TGM2 KO cells were labelled with a green or a deep-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated TGM2 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and deep-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, which was the case for all antibodies tested, except ab310333, ABCD_AI748 and A21184, we tested antibodies at using the dilutions listed below. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab109121* at 1/1000, ab109200** at 1/300, ab2386* at 1/1000, ab310333 at 1/500, ab421 at 1/1000, ABCD_AI748 at 1/1000, A21184** at 1/1000, ARP47471 at 1/500, ARP47472 at 1/250, AF4376 at 1/100, MAB4376* at 1/100, 3557** at 1/500, GTX111702 at 1/1000, 15100-1-AP at 1/300, 68006-1-Ig* at 1/500, MA5-32819** at 1/1000. Bars = 10 μm. Monoclonal antibody, *Recombinant antibody.\n\nIn conclusion, we have screened seventeen TGM2 commercial antibodies by western blot, and sixteen by immunoprecipitation, and immunofluorescence, comparing the signal produced by the antibodies in human A549 WT and TGM2 KO cells. Several high-quality antibodies that successfully detect TGM2 under our standardized experimental protocol can be identified. Researchers who wish to study TGM2 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.\n\nIn our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. Following the release of the antibody characterization, ab421 was removed from the manufacturer’s antibody catalog.\n\nThe authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway.27\n\nThe underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.28,29\n\n\nMethods\n\nThe standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a repository dedicated to openly sharing scientific research protocols, DOI: 10.21203/rs.3.pex-2607/v1.24\n\nCell lines used and primary antibodies tested in this study are listed in Table 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID.30,31", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for TGM2 (Protein-glutamine gamma-glutamyltransferase 2), https://doi.org/10.5281/zenodo.10819348. 28\n\nZenodo: Dataset for the TGM2 (Protein-glutamine gamma-glutamyltransferase 2) antibody screening study, https://doi.org/10.5281/zenodo.10927535. 29\n\nProtocol Exchange: A consensus for antibody characterization platform, https://doi.org/10.21203/rs.3.pex-2607/v1. 24\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.\n\nABIF consortium: Claire M. Brown and Joel Ryan.\n\nThank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.\n\nAn earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.10819348).\n\n\nReferences\n\nJeong EM, Lee KB, Kim GE, et al.: Competitive Binding of Magnesium to Calcium Binding Sites Reciprocally Regulates Transamidase and GTP Hydrolysis Activity of Transglutaminase 2. Int. J. Mol. Sci. 2020; 21(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanchan K, Ergülen E, Király R, et al.: Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity. Biochem. J. 2013; 455(3): 261–272. PubMed Abstract \| Publisher Full Text\n\nTatsukawa H, Furutani Y, Hitomi K, et al.: Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death. Cell Death Dis. 2016; 7(6): e2244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorand L, Graham RM: Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 2003; 4(2): 140–156. PubMed Abstract \| Publisher Full Text\n\nLiu S, Cerione RA, Clardy J: Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity. Proc. Natl. Acad. Sci. USA. 2002; 99(5): 2743–2747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAchyuthan KE, Greenberg CS: Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity. J. Biol. Chem. 1987; 262(4): 1901–1906. PubMed Abstract \| Publisher Full Text\n\nSingh US, Erickson JW, Cerione RA: Identification and biochemical characterization of an 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei. Biochemistry. 1995; 34(48): 15863–15871. PubMed Abstract \| Publisher Full Text\n\nMian S, el Alaoui S , Lawry J, et al.: The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression. FEBS Lett. 1995; 370(1-2): 27–31. PubMed Abstract \| Publisher Full Text\n\nPinkas DM, Strop P, Brunger AT, et al.: Transglutaminase 2 undergoes a large conformational change upon activation. PLoS Biol. 2007; 5(12): e327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIismaa SE, Mearns BM, Lorand L, et al.: Transglutaminases and disease: lessons from genetically engineered mouse models and inherited disorders. Physiol. Rev. 2009; 89(3): 991–1023. PubMed Abstract \| Publisher Full Text\n\nSzondy Z, Korponay-Szabó I, Király R, et al.: Transglutaminase 2 in human diseases. Biomedicine (Taipei). 2017; 7(3): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuo TF, Tatsukawa H, Matsuura T, et al.: Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways. J. Cell. Physiol. 2012; 227(3): 1130–1137. PubMed Abstract \| Publisher Full Text\n\nTatsukawa H, Fukaya Y, Frampton G, et al.: Role of transglutaminase 2 in liver injury via cross-linking and silencing of transcription factor Sp1. Gastroenterology. 2009; 136(5): 1783–95.e10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLesort M, Tucholski J, Miller ML, et al.: Tissue transglutaminase: a possible role in neurodegenerative diseases. Prog. Neurobiol. 2000; 61(5): 439–463. PubMed Abstract \| Publisher Full Text\n\nMin B, Chung KC: New insight into transglutaminase 2 and link to neurodegenerative diseases. BMB Rep. 2018; 51(1): 5–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJunn E, Ronchetti RD, Quezado MM, et al.: Tissue transglutaminase-induced aggregation of alpha-synuclein: Implications for Lewy body formation in Parkinson’s disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA. 2003; 100(4): 2047–2052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmid AW, Chiappe D, Pignat V, et al.: Dissecting the mechanisms of tissue transglutaminase-induced cross-linking of alpha-synuclein: implications for the pathogenesis of Parkinson disease. J. Biol. Chem. 2009; 284(19): 13128–13142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLai TS, Davies C, Greenberg CS: Human tissue transglutaminase is inhibited by pharmacologic and chemical acetylation. Protein Sci. 2010; 19(2): 229–235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilhelmus MM, van Dam AM , Drukarch B: Tissue transglutaminase: a novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases. Eur. J. Pharmacol. 2008; 585(2-3): 464–472. PubMed Abstract \| Publisher Full Text\n\nLai TS, Liu Y, Tucker T, et al.: Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries. Chem. Biol. 2008; 15(9): 969–978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, Ryan J, Biddle MS, et al.: Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023; 12: 12. Publisher Full Text\n\nCarter AJ, Kraemer O, Zwick M, et al.: Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24(11): 2111–2115. PubMed Abstract \| Publisher Full Text\n\nLicciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13(12): 1446–1459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, Ryan J, Bolivar SG, et al.: A consensus platform for antibody characterization (Version 1). Protocol Exchange. 2024. Publisher Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nBiddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12(12): 1344. Publisher Full Text\n\nAyoubi R, Fotouhi M, Alende C, et al.: Antibody Characterization Report for TGM2 (Protein-glutamine gamma-glutamyltransferase 2).2024. Publisher Full Text\n\nAyoubi R, Laflamme C: Dataset for the TGM2 (Protein-glutamine gamma-glutamyltransferase 2) antibody screening study. [Data set]. Zenodo. 2024. Publisher Full Text\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "289998", "date": "03 Jul 2024", "name": "Michael A Rieger", "expertise": [ "Reviewer Expertise Stem cells", "Cancer stem cells", "Single cell technologies", "TGM2 physiology in colorectal cancer and other cancers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 this study have tested 17 different commercially available antibodies against human Transglutaminase 2 (TGM2) on their specificity and feasibility for different biochemical and cell biological applications. They rigorously tested the antibodies for their suitability in Western blotting, immunoprecipitation and immune fluorescence. TGM2 is a multifunctional enzyme found in the cytosol and in the extracellular matrix. It functions as a glutaminase and a GDPase, depending on its conformation governed by the presence of Ca2+, thereby catalyzing peptide cross-linking and conducting signal transduction. TGM2 has been implicated in several diseases, such as celiac disease, cancer, neurodegenerative diseases, among others. Therefore, TGM2 has served as potential target for drug interference strategies in ongoing preclinical and clinical trials. The work presented here is of utmost interest for anybody working on the physiological and pathological function of TGM2, and provides solid data for selecting appropriate reagents to specifically and robustly detect TGM2 for research and diagnostic purposes. In general, work performed by the authors should be a prerequisite before using commercially available reagents (especially antibodies) to study biological effects and draw potentially wide-ranging conclusions which solely depend on the chosen diagnostic tool. Therefore, the manuscript here serves as a blueprint of how to test the validity of commercially available antibodies for several commonly used analytical methods. The authors provide here a most valuable resource of data, based on appropriately planned and conducted experiments. The manuscript is well written with high quality data representation, and the described methods are sound. The results shown here are of qualitative nature, and do not allow direct comparison of binding kinetics and sensitivity of the tested antibodies. Clear differences in the specificity, accuracy and suitability of the selected antibodies for the tested methods can be demonstrated, which will have consequences for evaluating and selecting TGM2 antibodies for future applications and studies, and for critical re-evaluation of findings from published studies. Last, vendors will decide on future continuation and/or recommendation of their products, based on these results. A few important minor points are still missing in this report, which the authors may want to address:\nThe sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "12032", "date": "18 Jul 2024", "name": "Kathleen Southern", "role": "Author Response", "response": "Dear Michael A. Rieger, The authors would like extend our gratitude for having taken the time to review this article and provide concrete feedback. Please see our response to your points (italicized) which we have responded to and/or addressed in a new version of the article, which will be submitted to F1000 shortly. We hope to have clarified any concerns or misunderstandings you previously had and believe the article is now ready for approval. Reviewer Comment: 1. The sensitivity and specificity of antibodies depend on the correctly titrated amount and concentration used for the respective assay. The authors refer to their general protocol on how to test antibodies for their applications, but they do not provide an easy-to-see overview of the used concentrations of the presented TGM2 antibodies for each application. The authors may want to extend the information shown in Table 2 for each application presented in figures 1-4. Author Response: We understand how the concentration of antibodies used in each application were not clearly accessible in the report. Based on our in depth procedure referred to in methods section ( https://protocolexchange.researchsquare.com/article/pex-2607/v1) and the dilutions presented in the Figure legends, we can assume the viewers can determine which concentration was used for the respective assays or are able to reproduce the experiment based on the information provided. Reviewer Comment: 2. TGM2 is expressed in different isoforms, and at least four isoforms have been robustly reported and studied. These isoforms differ in their c-terminal part of the protein, and only isoform 2 is significantly shorter with reduced molecular weight that can be seen by Western. It would be of interest for the research community to learn about the isoform specificity of the tested antibodies. The authors should at least provide a table with the immunogens/peptides used to generate these antibodies. In the long run, the consortium should consider to test isoform specificity by overexpressing TGM2 isoforms in null cell lines. Author Response: We understand this need and have added a column to table 2 to provide the readers with the immunogenic or peptide region the antibodies target, based on the information provided on the antibody manufacturer catalog. Reviewer Comment: 3. Along the same lines, the authors should check for TGM2 mRNA expression of the different isoforms in their wt cell line A549 by isoform-specific qPCR. They should also briefly mention the different TGM2 isoforms and their functions in the introduction and point out the current limitations of their study regarding the isoform specificity. Author Response: The authors are aware of that TGM2 isoforms exists, but that being said the purpose of this Data note is not to provide information regarding those isoforms but rather deliver a toolkit, to be used by researchers who wish to study TGM2 in relation to health and/or disease, by demonstrating which commercially available antibodies are of high-quality to be selected and used to enhance their research. Using these antibodies, these researchers can dive deeper and decipher whether the isoforms are present in the cell line. This level of detail is outside the scope of this initiative, which follows a platform (see link to protocol exchange preprint above) to validate antibodies by creating/obtaining knockout (KO) cell lines for a protein target and screening all commercial antibodies against that target head-to-head in key academic applications, comparing wild-type to KO cells." } ] }, { "id": "293962", "date": "15 Jul 2024", "name": "Nicoletta Bianchi", "expertise": [ "Reviewer Expertise cancer research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Editorial Team, I find this work extremely interesting. The authors tested the efficacy and specificity of commercial antibodies that can be used to study TGM2. Unfortunately, only one cell line and its KO were used, this is limiting, because the cell lines, especially cancer cells, can present different altered variants and/or different off-targets that could be recognized. At least the antibodies that are highly specific in this study could be tested on more cell lines. I believe that by contacting researchers who have developed TG2 KO lines or models, the authors could find a lot of willingness to collaborate. In any case, the manuscript is well done and of considerable applicative relevance and can help direct the researcher to a more appropriate choice of antibodies.\nHowever, for a better view of the applicability framework and before the indexing of this study, I suggest the inclusion of some antibodies from companies such as Zedira, which promotes a specific line of products for transglutaminase application, which are widely used by researchers working in the field of TGM2, as well as from Covalab. This would be of great interest and would greatly increase the impact of the work.\nMajor revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Minor revision: Keywords: typos errors, punctuation errors, “western” in capital letters, as well as when reported in the manuscript Introduction: “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Results and discussion: Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term.\nWith my best regards,\nNicoletta Bianchi\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "12071", "date": "30 Jul 2024", "name": "Kathleen Southern", "role": "Author Response", "response": "Dear Nicoletta Bianchi, Thank you for reviewing the article “A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence” recently published to the YCharOS gateway. In response to your major and minor revisions (italicized), the authors have edited the existing manuscript and will be submitting an updated version of the article shortly. We hope our response to your review clarifies any concerns or misunderstandings you may have previously had. Reviewer Comment - Major revision: The authors should implement the panel of antibodies including those widely used in publications on TGM2. Author Response - To address your major revision, in the newly submitted manuscript, we have included a column to Table 1 to indicated the number of times the tested antibodies were cited in published articles. The information is provided on CiteAb.com. Please visit the website if you’d like to replicate this study using other widely used antibodies that were not evaluated in this study. Reviewer Comment - Minor revision Introduction: “The proteins activity is dependent on cellular location, remaining inactive intracellularly, and becoming activated upon secretion”, the sentence is incorrect, because it is also active inside the cells depending on calcium release stimuli. “by a committee of industry, academic research”, which committee is this? must be specified. Some errors in the text. Author Response - The sentence regarding the protein’s activity being associated to cellular location has been removed. As for the committee of industry and academic researchers, we have clearly indicated that the list of participating members is mentioned in the competing interest section Reviewer Comment - Results and discussion: Authors should clarify the difference between the cell extract and lysate used. If they mean the same thing, it would be better to use the same term. Author Response - In the second paragraph of this section, we have removed the word “extracts” to prevent misinterpretation and have clarified that cells were collected as lysates (medium free) or on culture medium to test the antibodies by western blot." } ] } ]	1	https://f1000research.com/articles/13-481
https://f1000research.com/articles/13-849/v1	30 Jul 24	{ "type": "Research Article", "title": "Understanding the habitat vulnerability of Slums to COVID 19: Case of two megacities of India", "authors": [ "Sudha Panda", "Soumyendu Shankar Ray", "Soumyendu Shankar Ray" ], "abstract": "Background Urban slums are hotspots of infectious diseases like COVID-19 as was seen in the waves of 2020 and 2021. One of the primary reasons why slums are disproportionately affected is their location in inaccessible and uninhabitable zones, crowded and poorly ventilated living spaces, unsanitary conditions and common facilities (water taps, common toilets, etc.). Staying at home during pandemics is hardly an option for slum dwellers as it often means giving up work and even basic necessities.\n\nMethodology This paper aims to understand the habitat vulnerabilities of slums in the two Indian megacities of Pune and Surat which were the worst hit during both waves. The study is done at the level of wards, which is the smallest administrative boundary, taking the habitat vulnerability (congestion and access to basic services). To identify the explanatory variables which increase the vulnerability of slums to infectious diseases, literature study is done on the triggering factors which affect habitat vulnerability derived from common characteristics and definitions of slum.\n\nResults The aim of the research is to categorize the slums into 3 levels of risk zones and map them subsequently.\n\nConclusion This study will help in formulating a model to prioritize the allocation of sparse resources in developing countries to tackle the habitat vulnerabilities of the slum dwellers especially during health emergencies of contagious diseases like COVID-19.", "keywords": [ "Habitat vulnerability", "COVID-19", "urban slums", "congestion", "access to basic services", "risk exposure" ], "content": "Introduction\n\nIndia is among the foremost countries which were severely impacted by COVID-19 in the 2021 wave. It managed to dodge the severity of the impact of the 2020 wave by imposing a very rigid lockdown which just about managed to flatten the curve giving the government the time to scale up the public health system. But in future if we are to be better equipped to contain the transmission of a pandemic we have to primarily identify the risk factors that catalyze the disease spread in our urban realm and take appropriate steps to counter the same. It was observed that states with greater urban population, relatively higher population density, greater slum population, and a comparitively higher crowding in dwellings had a higher incidence of COVID-19. This was further intensified by comparatively poor medical facilities and lower expenditure on health infrastructure (Pathak et al., 2020). Social distancing which reduce exposure to COVID 19 is difficult to practice in slums which have crowded and congested living (Corburn et al., 2020; Friesen and Pelz, 2020). Besides space restriction and congestion, the lack of basic amenities like water supply, toilets, sewerage system, drainage facilities, solid waste collection, secure and adequate housing and existing poor medical conditions of the slum dwellers further reinforces their vulnerability to COVID-19 (Khatua, 2020).\n\nThe objective of this research is to obtain a critical understanding of the relationship between habitat vulnerability, health and slums so that risk exposure of slum dwellers can be quantified. The study is done at the smallest administrative level i.e. at ward level so that it is easy to identify the susceptible wards in a pandemic, enabling greater resources to be diverted to these places and taking preventive steps to counter the risk factors.\n\nSpread of the pandemic in urban centers was a global phenomenon. Cities with a larger population had a larger infection base and India was no exception with almost 40% of the cases in the 2020 wave being found in the 4 major metro cities of Delhi, Mumbai, Kolkata and Chennai which see a lot of international visitors. But the rapid spread of the contagion there onwards mainly happened where the urban concentration was more. The Census 2011 report states that one-third of India’s population lives in slums. Considering that the highly congested slums are breeding grounds for COVID 19 which thrives on proximity, it is imperative to understand the inherent habitat vulnerability of these slums to be able to propose an effective and long term policy planning to reduce these risk factors. Research has shown that in Mumbai, settlements have been built around industrial areas like garment factories where people stay in cramped spaces with eight people sharing a room surrounded by hazardous chemicals and machines (Parpiani M., 2020).\n\nDaily wage laborers and migrant labor constitute a major chunk of the slum dwellers (Ghosh et al., 2020). Social distancing is a safeguard against the spread of COVID-19 but it is a luxury which cannot be afforded by daily wage laborers living in slums (Courtemanche et al., 2020; Weill et al., 2020). For most slum dwellers it is important to continue livelihood activities which will ensure an income and subsequently food security than the possibility of contracting COVID-19.\n\nTo have a better understanding of the relationship between ‘Slums’, ‘habitat vulnerability’ and ‘health’ it is important to delve deeper into their definitions given by noted organizations.\n\nSlums\n\nThe term “slum” refers to squalid housing within urban areas that are overcrowded and have inadequate provision of basic services. UN-HABITAT definition for a slum household needs to satisfy one or more of the five conditions given below:\n\n- Temporary housing that does not give protection against extreme climate conditions;\n\n- Inadequate dwelling space;\n\n- Availability of affordable, sufficient and safe water;\n\n- Provision of a private or public toilet and\n\n- Security of tenure (UN-Habitat 2003a; 2003b; 2006/7).\n\nWorld Health Organization (WHO) recommends 2m physical distancing as a preventive against COVID 19 spread which is next to impossible in a slum household where 5-6 persons are packed in a 25-30 square meters dwelling with poor ventilation which is further aggravated due to cooking methods which rely on coal or wood as fuel. Hygiene and access to clean water is a critical parameter for precaution against COVID-19 but with large number of people dependent on municipality tankers for water and very low ratio of private toilets it is very difficult to practice the hand washing and hygiene norms suggested by WHO.\n\nWe take a comparative look in Table 1 on various definitions and parameters (Legality, Density, Housing, Water and Sanitation) by various noted national and international organizations to understand their commonality.\n\nVulnerability and risk exposure\n\nThe term “Vulnerability” refers to certain inherent and inbuilt set of conditions that maintain people in a perilous and hazardous conditions. In the context of susceptibility to health, it implies situations leading to risk to diseases. It can also be defined as “the degree to which a system, subsystem or system component is likely to experience harm due to exposure to a hazard, either to a perturbation or stress or stressors” (Turner et al., 2003). Risk is the chance (or possibility) that the hazard will reoccur. Vulnerability is a function of exposure, sensitivity and adaptive capacity of the system (Cutter, 1996).\n\nHealth vulnerability of Slums to COVID 19 due to habitat conditions\n\nThe definition of vulnerability in the context of health leads us to understand the inherent structural conditions in slums that makes the slum dwellers prone to contagious diseases like COVID-19. It mainly spreads through contact, hence the health vulnerability in slums is mainly due to the inability to practice physical distancing and other conditions which aid the disease transmission once the disease has been contracted. The literature study revealed that the health vulnerability parameters which exist in slums can be clubbed into two types of factors\n\n1. Socio Economic Conditions\n\n2. Habitat Condition\n\nTable 2 (below), clubs all the factors derived from literature study into the above headings to later filter out the factors can be controlled or improved.\n\n\nMethods\n\nThe study was conducted with an objective to understand the existing habitat vulnerability which is present in slums which aggravates the susceptibility of the slum dwellers to contagious diseases like COVID-19. In order to identify these explanatory variables, the characteristics which define a slum given by various organizations are studied and superimposed on the habitat and socio-economic factors identified by previous literature which are considered responsible for increasing the vulnerability of slum dwellers. Data is collected about the common parameters (congestion and access to basic services) from sample cities of Surat and Pune wherein a ward (smallest administrative border in the city) is the unit of comparison.\n\nTo derive the risk intensity, the raw data is transformed into relative values to normalize all values so that they range from 0 to 1. After giving weights to the indicators a linear summation is done to obtain a composite value for the risk exposure and depending on the range classified into high, medium or low. When this risk exposure value is referenced on the ward map of the city, a lucid and graphic picture emerges for policy planners while prioritizing the allocation of scarce resources to areas which demand urgent attention and to be fully prepared in the event of a disaster like COVID-19. Figure 1 (below) shows the complete research steps followed to achieve this end.\n\nPune and Surat have been selected for testing this model because they are contrasting cases of cities in the way they have dealt with slum issue. Pune has 40.56% of its population living in slums, whereas Surat has only 19.24% of its population living in the slums. The low slum population in Surat is due to the aggressive implementation of slum re-location and development programs after the plague in 1992.\n\nPune\n\nPune municipal corporation (PMC) is responsible for the provision of basic services in slums under the Maharashtra Slum Improvement and Clearance Act of 1971. As of 2011, Ghole Road, Dhole Patil Road and Sangamwadi ward shows the maximum percentage of slum population (between 40-50%). The least number of slums population is found in Bibvewadi, Kasba and Dhankawadi wards (less then 5%). Tilak Road ward and Karve Road have maximum number of declared slum i.e. about 13% and 12% respectively.\n\nA study of the trend between slum population percentage and COVID-19 infection rate during both waves of 2020 and 2021(as seen in Figures 2 and 3) shows that there’s no particular relationship that would imply a higher population in slums having a higher infection rate.\n\n(Source: Pune Municipal Corporation, 2020).\n\n(Source: Mohol, 2021).\n\nSurat\n\nSlums in Surat constituted 27.5 percent of the city’s population in 1992. However, after 1992, the slum growth rate has decreased from an annual average of 14.6 percent in 1992 to an annual average of 1.46 percent in 2001.\n\n(Source: Surat Municipal Corporation, 2020).\n\nIn Surat too, a study of the trend between slum population percentage and COVID-19 infection rate during both waves of 2020 and 2021(as seen in Figures 4 and 5) shows the same results as in Pune. There’s no particular relationship that would imply a higher population in slums having a higher infection rate.\n\n(Source: Surat Municipal Corporation, 2021).\n\nEmpirical Model\n\nBased on the habitat vulnerability parameters obtained from the literature study and superimposing on the parameters which define a slum (given by noted organizations) it was seen that ‘Congestion’ and ‘Lack of access to Basic Services’ come out as the most significant parameters responsible which create vulnerability of slums to COVID-19. The two indicators selected under ‘Congestion’ were ‘Population’ and ‘Density’ (persons per unit square km in slums). The four indicators selected under ‘Lack of access to Basic Services’ were ‘Percentage population with access to water supply’, ‘Percentage population using private toilet’, ‘Percentage population using common toilet’ and ‘Percentage population with near accessibility to health facility’. Expert opinion on the weightage which should be given to the final six indicators were 0.25 to each of the two ‘Congestion indicators’ and 0.125 to each of the four ‘Lack of access to Basic Services’ indicators.\n\nHence the linear model obtained was\n\nRisk Exposure of a slum = α1 + β1 Congestion factors (f(‘Population’, ‘Density’)) + Lack of access to Basic services (f(‘Percentage population with access to water supply’, ‘Percentage population using private toilet’, ‘Percentage population using common toilet’ and ‘Percentage population with near accessibility to health facility’)) + εi\n\nTrue values of indicators for sample cities of Pune and Surat are seen in Tables 3 and 4 (below)\n\n\nResults and discussion\n\nAs can be seen in the linear model above measuring risk exposure of the slums the indicators for “congestion” and “access to basic services” have different measurement units (persons, persons/sqkm and percentages etc). However, to make these indicators comparable they need to be transformed into a standard form. Hence, each of the indicators included in the analysis, is normalized using the actual, minimum and maximum risk threshold values. The value of the normalized relative indicator varies between 0 to 1 and is calculated thus:\n\nFor some indicators, a higher score is equivalent to a higher risk (as in the congestion factors), whereas for other indicators, a higher score might imply lower risk (as in percentage access to water and individual toilet facilities). For the scores to be formulated according to higher the value the lower the risk (as in percentage access to water and individual toilet facilities), the values are transformed into negative values.\n\nTo obtain a composite value of the risk\n\nWhere Ri the overall score of Risk i and Sil the Relative indicator value i for criterion j of which wj is the weight.\n\nCalculating the composite values of the Risk exposure across the different wards in Pune and Surat, the following values are obtained as seen in Tables 5 and 6.\n\nTransforming these numeric values into a more comprehensive picture, a range of > 0.20 is taken as high, 0 to 0.20 is taken as medium and less than 0 is taken as low. Finally, to represent the values graphically, it is graphically represented on the ward map of the cities as seen in Figures 6 (Pune) and 7 (Surat).\n\n(Source: Authors, 2023).\n\n(Source: Authors, 2023).\n\nA major part (9 out 15 wards) of the Pune slums fall in the high risk zone. Only 3 are in the medium risk zone and 2 are in the low risk zone. With almost 40.56% of the Pune population living in slums, this spells trouble for the entire city as slums are the hotspots from where the contagion starts spreading.\n\nThe picture is much brighter in Surat, in spite of the rapid influx of migrants, as only 3 wards are in high risk zone. This is due to the proactive measures taken by Surat Municipal Corporation (SMC) in 1994 when they took aggressive and rigorous measures after the 1994 plague. However, in spite of the concentrated efforts of the Surat municipality there were open areas in the south and south eastern parts of the city where due to rural migration slums developed again.\n\n\nConclusion\n\nThe aim of the paper was to find a method to quantify the risk exposure to pandemics in slums through a critical understanding of the relationship between habitat vulnerability, health and slums which was done by taking a case study of 2 cities of Pune and Surat during the COVID-19 waves of 2020 and 2021. By concentrating on the parameters which define a slum and are simultaneously the triggering factors for the spread of a pandemic (as revealed by the literature study), a method is devised to quantify the risk exposure.\n\nIt is important to find both immediate and long term solutions to the problem. COVID 19 has cemented the fact this will not be the first or last contagious disease which the world will witness in a long time but we need to be better prepared to contain such pandemics and the first step lies in curtailing its spread in the hotbeds i.e. the slums of urban centers. By identifying the critical slums the study has found a method for quantifying the risk exposure due to habitat conditions which will help to prioritize the allocation of scarce resources in emergency times and also for long term slum redevelopment planning.\n\nAs an immediate measure, access to provision of basic amenities should be prioritized in the times of pandemics by water tanks, mobile sanitizing centers/hand washing stations, portable toilets at critical points which can be done under Swachh Bharat Abhiyaan or Clean India Mission. To incentivize the collection of solid waste, youth in the slum can be encouraged by giving a bag of groceries in exchange for a bag of solid waste. The bags of solid waste obtained can be recycled by the slum dwellers and be a source of employment generation. This can be managed by slum emergency planning committees which will also provide mobile health clinics to carry out testing, diagnosis and immediate treatment. These mobile clinics should be able to maneuver through the existing narrow streets in the slums and be well equipped with equipment for the treatment of COVID-19 and common ailments in urban slums, which are usually diarrhea and respiratory infections. The long term measures should be relocation or redevelopment of slums in a phased manner providing well ventilated housing having all the basic amenities like water, sanitation, drainage, toilets, access to health facilities.\n\nThe University has granted consent to participate and publish.\n\n\nAuthor’s contribution\n\nPanda S: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, writing – Original Draft Preparation, writing – Review & Editing; Ray SS: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, writing – Original Draft Preparation, Writing – Review & Editing;\n\n\nEthical approval and consent for third party data\n\nThe Ethics Committee of the School of Architecture and Planning, KIIT University on 6th June,2024 waived the need for ethics approval and the need to obtain consent for the collection, analysis and publication of the data as it has been obtained from freely available online content which has been properly referenced and cited.\n\n\nConsent to participate\n\nAll authors have given consent to participate.\n\n\nConsent to publish\n\nAll matter in paper is original work and not published elsewhere. There is consent to publish.", "appendix": "Data availability statement\n\n1. Figshare: Ward wise Covid 19 Positive Cases in Pune City on May 29, 2021. https://doi.org/10.6084/m9.figshare.26196443 (Panda & Ray, 2024a).\n\nThe project contains the following underlying data:\n\n• Figure 1.jpeg (Covid 19 positive cases in Pune on May 29, 2021).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n2. Figshare: Ward wise Covid 19 Positive Cases in Pune City on 11 May, 2020. https://doi.org/10.6084/m9.figshare.26196449 (Panda & Ray, 2024b).\n\nThe project contains the following underlying data:\n\n• Figure 2.jpeg (Covid 19 positive cases in Pune on May 11, 2020).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n3. Figshare: Ward wise Covid 19 Positive Cases in Surat City on 1 May, 2021. https://doi.org/10.6084/m9.figshare.26196455 (Panda & Ray, 2024c).\n\nThe project contains the following underlying data:\n\n• Figure 3.jpeg (Covid 19 positive cases in Surat on May 1, 2021).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n4. Figshare: Ward wise Covid 19 Positive Cases in Surat City on 31 July, 2020. https://doi.org/10.6084/m9.figshare.26196464 (Panda & Ray, 2024d).\n\nThe project contains the following underlying data:\n\n• Figure 3.jpeg (Covid 19 positive cases in Surat on July 31, 2020).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe are thankful to the administrative support provided by our University.\n\n\nReferences\n\nAyeb-Karlsson S, Kniveton D, Cannon T: Trapped in the prison of the mind: Notions of climate-induced (im) mobility decision-making and wellbeing from an urban informal settlement in Bangladesh. Palgrave Commun. 2020; 6(1). Publisher Full Text\n\nAyeb-Karlsson S: ‘When we were children we had dreams, then we came to Dhaka to survive’: urban stories connecting loss of wellbeing, displacement and (im)mobility. Clim. Dev. 2020; 13(4): 348–359. Publisher Full Text\n\nCorburn J, Vlahov D, Mberu B, et al.: Slum Health: Arresting COVID-19 and Improving Well-Being in Urban Informal Settlements. J. Urban Health. 2020; 97(3): 348–357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCourtemanche C, Garuccio J, Le A, et al.: Strong social distancing measures in the United States reduced the COVID-19 growth rate. Health Aff. 2020; 39(7): 1237–1246. PubMed Abstract \| Publisher Full Text\n\nCutter SL: Vulnerability to environmental hazards. Prog. Hum. Geogr. 1996; 20(4): 529–539. Publisher Full Text\n\nEzeh A, Oyebode O, Satterthwaite D, et al.: The history, geography, and sociology of slums and the health problems of people who live in slums. Lancet. 2017; 389(10068): 547–558. PubMed Abstract \| Publisher Full Text\n\nFriesen J, Pelz PF: COVID-19 and Slums: A pandemic highlights gaps in knowledge about urban poverty. JMIR Public Health Surveill. 2020; 6(3): e19578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeorge CE, Inbaraj LR, Chandrasingh S, et al.: High seroprevalence of COVID-19 infection in a large slum in South India; what does it tell us about managing a pandemic and beyond? Epidemiol. Infect. 2021; 149: e39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhosh S, Seth P, Tiwary H: How does Covid-19 aggravate the multidimensional vulnerability of slums in India? A Commentary. Soc. Sci. Humanit. Open. 2020; 2(1): 100068. Publisher 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\nGupta K, Arnold F, Lhungdim H: Health and Living Conditions in Eight Indian Cities. Calverton, Maryland, USA: National Family Health Survey (NFHS-3), India, 2005-06. Mumbai: International Institute for Population Sciences; 2009. ICF Macro. Reference Source\n\nKhatua S: Density, distancing, informal settlements and the pandemic. Econ. Polit. Wkly. 2020; 55(20). Accessed October 11, 2022. Reference Source\n\nMahajan S: Housing Study for Pune Municipal Corporation (2009-2010). Maharashtra State Housing and Action League; 2010. Reference Source\n\nMinistry of Housing and Urban Poverty Alleviation: Report of the Committee on Slum Statistics/Census. New Delhi, India: Government of India, Ministry of Housing and Urban Poverty Alleviation; 2008. Reference Source\n\nNolan LB, Bloom DE, Subbaraman R: Legal status and deprivation in India’s urban slums: an analysis of two decades of national sample survey data, PGDA Working Paper No. 135 Harvard University Program on the Global Demography of Aging Working Paper Series. 2017.2017. Reference Source\n\nPanda S, Ray SS: Ward wise Covid 19 Positive Cases in Pune City on May 29, 2021. figshare. Figure. 2024a. Publisher Full Text\n\nPanda S, Ray SS: Ward wise Covid 19 Positive Cases in Pune City on 11 May, 2020. figshare. Figure. 2024b. Publisher Full Text\n\nPanda S, Ray SS: Ward wise Covid 19 Positive Cases in Surat City on 1 May, 2021. figshare. Figure. 2024c. Publisher Full Text\n\nPanda S, Ray SS: Ward wise Covid 19 Positive Cases in Surat City on 31 July, 2020. figshare. Figure. 2024d. Publisher Full Text\n\nParpiani M: Fighting fires: migrant workers in Mumbai. Econ. Polit. Wkly. 2020; 55(14). Reference Source\n\nPathak PK, Singh Y, Mahapatro SR, et al.: Assessing Socioeconomic vulnerabilities related to COVID-19 risk in India: A State-Level Analysis. Disaster Med. Public Health Prep. 2020; 16(2): 590–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPune Municipal Corporation: Check area wise covid patients in Pune city. Tally reaches 3105 in Pune district. Punekar News. 2020, May 11. Reference Source\n\nMohol MK: [@mohol_murlidhar]. Ward wise Covid-19 Positive Cases in Pune City. [Tweet]. Twitter.2021, May 29. Reference Source\n\nRaju E, Ayeb-Karlsson S: COVID-19: How do you self-isolate in a refugee camp? Int. J. Public Health. 2020; 65(5): 515–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaju E: Editorial. Disaster Prev. Manag. 2020; 29(4): 421–423. Publisher Full Text\n\nRaju E, Dutta A, Ayeb-Karlsson S: COVID-19 in India: Who are we leaving behind? Prog. Disaster Sci. 2021; 10: 100163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSclar ED, Garau P, Carolini G: The 21st century health challenge of slums and cities. Lancet. 2005; 365(9462): 901–903. PubMed Abstract \| Publisher Full Text\n\nSubbaraman R, O’brien J, Shitole T, et al.: Off the map: the health and social implications of being a non-notified slum in India. Environ. Urban. 2012; 24(2): 643–663. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSurat Municipal Corporation: Zonewise confirmed cases. Surat Smart City: 2020, July, 31. Reference Source\n\nSurat Urban Development Authority: Surat City development Plan (2006-2012).2006-12. Reference Source\n\nSurat Municipal Corporation: Zonewise confirmed cases. Surat Smart City: 2021, May, 01. Reference Source\n\nTurner BL, Kasperson RE, Matson PA, et al.: A framework for vulnerability analysis in sustainability science. Proc. Natl. Acad. Sci. USA. 2003; 100(14): 8074–8079. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUN-HABITAT: “The Challenge of Slums: Global Report on Human Settlements”. 2003b. “Slums of the World: The face of urban poverty in the new millennium?”. 2006/7. “State of the World’s Cities 2006/2007.”. Nairobi, Kenya: 2003.\n\nWasdani KP, Prasad A: The impossibility of social distancing among the urban poor: the case of an Indian slum in the times of COVID-19. Local Environ. 2020; 25(5): 414–418. Publisher Full Text\n\nWeill JA, Stigler M, Deschenes O, et al.: Social distancing responses to COVID-19 emergency declarations strongly differentiated by income. Proc. Natl. Acad. Sci. USA. 2020; 117(33): 19658–19660. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "314475", "date": "29 Aug 2024", "name": "Natraj Kranthi", "expertise": [ "Reviewer Expertise Geospatial modeling for urban planning applications", "tenure security for the urban poor", "and inclusive planning" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary of the Paper - The paper titled ‘Understanding the habitat vulnerability of Slums to Covid 19: Case of two megacities of India’ aims to categorize the slums into three levels of risk zones for mapping. For this purpose, ward wise habitat vulnerabilities of slums in the two Indian megacities of Pune and Surat were analysed. Definitions given by UN-Habitat and WHO were reviewed and vulnerability parameters ‘congestion’ with two indicators and ‘lack of access to basic services’ with four indicators were identified. Risk exposure was measured based on the normalized true values. Risk was calculated and categorized as high (>0.2), medium (0-0.2) and low (<0). Accordingly these ward wise risks maps were done. The paper concludes by suggesting the importance for immediate and long term solutions Observations\n\ni. Definitions - The definitions of slums given by the international organizations like UN-HABITAT and WHO were reviewed. However, the Indian definitions of slums such as (i). Notified slums (as per the Slum Act 1956), (ii). Identified slums (as per the Census of India) and (iii). Recognized slums (that are not formally notified but recognized by the authorities) were not reviewed. As the study is conducted in the Indian cities of Pune and Surat, it becomes imperative to review these definitions as well besides the international definitions. ii. Identification of vulnerability parameters – Based on the review of both the international and national definitions, the habitat vulnerability parameters and indicators are to be identified. The number of such parameters may increase accordingly. Therefore, there is a need to augment the literature review in this regard. iii. Identification of indicators - Under ‘congestion’, two indicators ‘Population’ and ‘Density’ were identified. However the current literature such as Namperumal et al (2022) reveals a direct link between builtup density and positive Covid cases. Therefore, it is suggested to cite the current literature and consider ‘Builtup density’ also as one of the indicators under ‘congestion’. iv. Cities as study area – In the abstract, it is mentioned that two Indian megacities Pune and Surat were considered as they were the worst hit during both the waves. However no adequate data or reference is cited to substantiate this. In the background, it is mentioned as ‘40% of the cases in the 2020 wave being found in the 4 major metro cities of Delhi, Mumbai, Kolkata and Chennai’. If it is so then why these cities were not considered for the study? v. Study at the ward level – The study was carried out at the ward level and not at the slum level, while the title mentions as vulnerability of slums. The paper does not specify the number of slums considered in each of the wards. vi. True values – It is not clear as to how the true values were collected/ calculated (see Tables 3 and 4). The following questions need to be answered: How many slums were considered and based on what criteria? What is the sampling type? What is the sample size? Therefore, there is a need to improve the study design to make it more technically sound. vii. Data –Open source data for covid 19 risk zones (such as covid dashboard of sociometrik website) are available. Official data are available from the covid dashboard of the Ministry of Health and Family Welfare, Government of India. Therefore, there is a need to refer to such source data to ensure reproducibility. viii. Data presentation - Figures 4 and 5 show the ward wise relation between % Slum Population and Covid 19 infection rate. However the graphs shown are not ward wise but zone wise (south, west, north, etc.). Figures 2 & 3 and Table 3 has the ward names only but not the ward numbers, while the maps show only the ward numbers (Figure 6) and not the ward names. Therefore, there is a need to improve the data presentation and analysis. ix. Methods – Need to clarify the criteria for using/ adopting the linear model. Relevant literature can be reviewed for adopting such models for the purpose of calculation/ analysis. Therefore, there is a need to improve the method. x. Basis for ranges– Need to clarify the basis for considering the ranges (> 0.20 is taken as high, 0 to 0.20 is taken as medium and less than 0 is taken as low). Is there any literature to substantiate these ranges? Therefore, there is a need improve the method and statistical analysis. xi. Conclusion – In the abstract, the aim is to categorize the slums into 3 levels of risk zones. However in the conclusion, the aim is mentioned as to find a method to quantify the risk exposure to pandemics in slums. Also, It is not clear on what basis the immediate and long term solutions are suggested. Therefore, the conclusions do not fully reflect the results. Refer https://sociometrik.shinyapps.io/covid19riskzones/ https://covid19dashboard.mohfw.gov.in/ and [1]\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "326376", "date": "28 Sep 2024", "name": "Partha Sarathi Mishra", "expertise": [ "Reviewer Expertise Architectural Design", "Heritage Assessment", "Statistical Analysis in Architecture and Planning", "Multi-Criteria Decision- Making Analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: This paper makes a valuable contribution by examining the habitat vulnerability of slums to COVID-19 in two major Indian cities, Pune, and Surat. The authors develop a quantitative risk assessment model based on congestion and access to basic services to categorize slum areas into high, medium, and low risk zones. The visual mapping of these risk zones provides an intuitive understanding of vulnerability patterns, which can inform targeted interventions. Major strengths: - Addresses a critical and timely topic of slum vulnerability to pandemics - Develops a quantitative risk assessment model that can be applied to other urban areas - Provides clear visual representation of risk zones through ward-level mapping - Offers both immediate and long-term recommendations for reducing vulnerability - Uses data from two different cities to demonstrate broader applicability of the approach Minor revisions suggested: 1. Consider including a brief overview of Indian definitions of slums to complement the international definitions provided. This would strengthen the contextual relevance of the study. 2. Provide some additional background on why Pune and Surat were selected as case studies, perhaps with a sentence or two of data on their COVID-19 impacts. 3. Add a few more details on data collection methods and sources in the methodology section to enhance reproducibility. 4. Ensure consistency in how ward data is presented across all tables and figures for clarity. 5. Simple Additive Weighting Method is mentioned in this paper. However, the weighting method and their implications are not available in this paper. Please clarify on this issue 6. Briefly explain the rationale for the risk score thresholds used (high >0.20, medium 0-0.20, low <0) to help readers interpret the results. 7. Consider adding a short limitations section to acknowledge any constraints of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "326369", "date": "23 Oct 2024", "name": "Deepashree Choudhury", "expertise": [ "Reviewer Expertise Built Environment", "Architecture", "Urban Design", "Urban Studies", "Research methodology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article investigates the vulnerability of slum habitats to COVID-19 in two Indian megacities. It seeks to understand how the unique living conditions in these informal settlements increase exposure to the virus and influence the overall pandemic response. The study addresses an important topic, given the disproportionate impact of the pandemic on marginalized urban populations. However, while the research is insightful, there are a few areas where the methodology, data presentation, and discussion could be strengthened to ensure clarity and replicability. 1. Presentation and Literature Citations\n\nThe paper is well-written, but certain sections, particularly in the introduction and conclusion, could be enhanced by integrating more recent studies on the impact of COVID-19 on slum populations globally and within India. The discussion of the slum environment in relation to pandemic spread is critical. Suggestions: (i)Strengthen the introduction by adding more recent studies on slum vulnerability to infectious diseases and specifically COVID-19. (ii)Provide proper citations for claims related to the broader impacts of COVID-19 on slums beyond the two cities studied. (iii)Clarify how this study builds upon or diverges from other research in this domain.\n2. Study Design and Technical Soundness The study provides valuable insights, but the rationale for choosing these two specific megacities (Pune and Surat) needs to be expanded. The authors only say’ Pune and Surat have been selected for testing this model because they are contrasting cases of cities in the way they have dealt with slum issues. Pune has 40.56% of its population living in slums, whereas Surat has only 19.24% of its population living in the slums. The low slum population in Surat is due to the aggressive implementation of slum re-location and development programs after the plague in 1992.‘\n\nThe authors do not fully explain whether the findings can be generalized to slums across India or if they are unique to these particular cities.\n\nSuggestions: (i) Add more robust justification as to why Pune and Surat were chosen as case studies over other cities that also have significant slum populations, such as Kolkata or Chennai. (ii)Discuss whether these findings are representative of slum vulnerability across India or only applicable to the megacities studied.\n3. Details of Methods and Analysis\nProviding a clear breakdown of how data was collected and Specifying the criteria used to select slums in these cities for the analysis will help enrich this research. A few questions come to the mind of the reader like, whether any specific sampling method was used, and whether the research involved any first-hand data collection(through interviews with slum dwellers or government officials) ? Were the selected case studies representative of slums across the megacities?\n4. Statistical Analysis and Interpretation\n\n(i)The authors state that expert opinions were used to assign weightage to the final six indicators—0.25 for each of the two ‘Congestion indicators’ and 0.125 for each of the four ‘Lack of Access to Basic Services’ indicators. However,an elaboration on how this expert opinion was collected (the process and criteria used to select experts, as well as the methods employed (e.g., surveys, interviews, consensus-building techniques) will highly aid in making the article more comprehensible and in validating the weightage assigned to the indicators. (ii) The authors mention that numeric values were transformed into categories (high, medium, low) based on specific ranges. Detailing out how the risk exposure benchmarks were derived, for the ranges designated as high, medium, and low will help in establishing the validity of the conclusions drawn.\n(iii)\n\nIn Tables 5 and 6, it is suggested that indicators be assigned nomenclature (e.g., Si1, Si2, etc.) to facilitate clearer comprehension and reference throughout the text. This labeling would enhance the readability of the tables and make it easier for readers to track the indicators discussed.\n\n5. Conclusions While the conclusions align with the overall findings, they are somewhat overstated, particularly regarding the implications for policy. The authors suggest wide-ranging solutions to slum vulnerability, but these are not fully supported by the data presented. The discussion would benefit from a more cautious interpretation of the findings, especially given the limitations of the study. Suggestions: (i)Acknowledge the limitations of the study more explicitly in the conclusions, especially in terms of data limitations and the representativeness of the findings. Provide more grounded, evidence-based policy recommendations that are supported by the specific data from the study. (ii)Consider offering suggestions for future research that could address gaps identified in this study, particularly in terms of broader applicability to other cities and long-term vulnerability assessment. (iii)separate out recommendations from the conclusion section and present them under a different head.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-849
https://f1000research.com/articles/12-1535/v1	30 Nov 23	{ "type": "Research Article", "title": "Current challenges in teaching HAIs-PC in nursing education in Vietnam and Cambodia: a qualitative study", "authors": [ "Anh Tuan Truong", "PrevInf Group: Capacitating Asia's Nursing Students on Innovative and Sustainable Prevention and Control of Healthcare-associated Infections" ], "abstract": "Background: There is an insufficient understanding of factors that impede nursing students’ learning of healthcare-associated infection prevention and control in developing countries. This study aimed to explore current challenges in healthcare-associated infection control and prevention education in the nursing curriculum in two Vietnamese and two Cambodian universities.\nMethods: Exploratory research was conducted through consultation of education programs and a qualitative study design utilizing interviews and focus group discussions. Data collection was conducted through interviews with university board members and focus group discussions with lecturers and tutors. The data were analyzed by using content analysis methods.\nResults: The research results indicated that there were three generic themes of challenges in teaching HAIs-PC in nursing education in Vietnam and Cambodia. They were Implementation of healthcare associated infections prevention and control education into nursing curriculum, Positive aspects fostering healthcare associated infections prevention and control learning, Negative aspects hindering healthcare associated infections prevention and control learning.\nConclusions: The study results provided evidence of challenges in healthcare associated infections prevention and control education in some Asia higher education institutions. To improve professional safety, universities should pay more attention to developing appropriate teaching methods for healthcare-associated infections prevention and control education to improve students’ practice outcomes.", "keywords": [ "healthcare associated infections prevention and control", "nursing education", "challenges", "Vietnam and Cambodia." ], "content": "Introduction\n\nHealthcare-associated infections (HAIs) are infections people get while receiving health care in any healthcare facility, including hospitals, ambulatory surgical centers, and long-term care facilities.1 Bacteria, fungi, viruses or other, less common pathogens can cause HAIs. HAIs are often acquired from hospitals after 48 hours or more of admission or within 30 days after having received health care. The World Health Organization (WHO) report shows a high rate of Antimicrobial Resistance and estimates 10 million deaths per year due to HAIs in 2050.2 The various consequences of HAIs for patients and the health system are included e.g., the increased morbidity and mortality of patients, prolonged average hospital stay from 7 to 15 days, and increased use of antibiotics leading to a resistance of microorganisms.\n\nThe incidence of HAIs is higher in developing countries compared to developed countries.2,3 Therefore, the increasing prevalence of HAIs in Asian countries increases the costs of healthcare organizations and the morbidity and mortality of patients in already low-income countries.4 The overall prevalence of HAIs in developing countries is around 17.0%, in which the rate of infections acquired in intensive care units is at least two to three times higher than in high-income countries.5\n\nIn Vietnam, previous findings indicated that the rate of hospital infections was around 6%, of which respiratory tract infections accounted for the highest rate at 56%. The risk factors associated with HAIs such as age (18-30 years old), gender (male), length of hospital stay (9 days), and invasive procedures.6 The prevalence of HAIs in the adult ICU accounted for around 23-30%.7,8 In addition, the rate of antibiotic resistance in Vietnam is a significant concern. Tran and colleagues (2019) indicated that the rate of gram-negative bacteria infection in inpatients is high. Infectious diseases accounted for 25% of hospitalizations and had a high rate of mortality (12%).9\n\nOn the other hand, in Cambodia, the most-reported ones were respiratory tract infections accounting for 53%.10 The study conducted at the National Hospital for Children indicated that HAIs incidence was around 14%, and the incidence of HAIs in ICU was 50%.10 The various rates of HAIs in different Asian countries and hospitals could be due to different morbidity patterns, treatment protocols, level of development of health systems, or HAIs prevention and control (HAIs-PC). Moreover, the different operational definitions of HAIs adopted in the studies and the low notification and monitoring of these events can also be the reason for the variation in HAIs rates across countries.11\n\nEven though prevalence statistics regarding HAIs are not sufficiently reported, there are many problems, which hinder systematic HAIs-PC work in developing countries.12 The awareness of the healthcare workers is not adequate, and at the same time, the lack of support resources such as education and medical equipment leads to a high percentage of HAIs. In Vietnam, according to Ngo (2018), healthcare workers reported challenges related to HAIs including poor resources, awareness, and patient overload.6 In 2015, WHO emphasized the importance of strengthening HAIs-PC by improving awareness among healthcare workers and patients.13 Governments of different countries indicated that it is necessary to promote healthcare, thereby consolidating and perfecting the system of surveillance, warning, and proactive disease prevention; establishing units of disease control and prevention.13\n\nThe provision of quality education is essential for the training and development of the appropriate competencies, knowledge, and skills of healthcare workers, which enables them to provide safe and quality healthcare. In Asian countries, healthcare universities gradually introduced HAIs-PC education programs for students at all levels.14 These programs demonstrated certain effectiveness of education in raising awareness and reducing the rate of HAIs. In Cambodia, following the WHO’s global action plan, efforts have been made to enhance the quality of Cambodia's health institutions’ conditions, including infection prevention and control practices.13 Moreover, the research evidence recommended that hospitals and healthcare universities should provide appropriate HAIs-PC education to students, patients, and healthcare providers, especially, nursing students at the very beginning of their bachelor program.15,16\n\nIn Asian countries, the HAIs-PC education program currently differs between universities and countries in contents and form of practice training, as well as in assessment and recognition.27 These disparities have led to difficulties in evaluating the effectiveness of the education program in improving nursing students’ knowledge and competence in this field. Moreover, there is insufficient scientific research to evaluate the perception of lecturers and learners about HAIs-PC education in developing countries. The research aim thus was to explore current challenges in HAIs-PC education in nursing curriculum in two Vietnamese and two Cambodian universities.\n\n\nMethods\n\nA qualitative study was conducted through interviews and focus group discussions among managers, nursing faculty teachers, and clinical tutors.\n\nThe study was conducted in four Higher Education Institutions (HEIs) in Vietnam and Cambodia. These institutions offer nursing education at levels of an associate degree in nursing (ADN), Bachelor of Nursing Science (BNS), and Master of Nursing Science (MNS).\n\nThe participants of the study were invited based on the inclusion criteria: (1) working in the university or in the hospital and teaching contents related to HAIs-PC; (2) having at least a bachelor’s degree; (3) having at least five years’ experience in teaching contents related to HAIs-PC. In each institution, the participants of the study were divided into three groups. The first group consisted of managers, coordinators of scientific boards, and the heads of the nursing department. The second group consisted of nursing faculty teachers who directly teach module of HAIs-PC to nursing students. The third group consisted of clinical tutors who provided instructions for students in the hospitals. They were full-time faculty teachers in the universities or full-time nurses in the hospitals.\n\nThere were four instruments used for data collection developed by the research team, including one quantitative and three qualitative instruments.\n\nA 26-item record collection instrument was used to collect the indicators of HAIs-PC education in each institution about the overall duration of the specific curriculum, the number of credits for each module including theory and practice, and the lecturer’s and the student’s information in the nursing curriculum. Especially, the HAIs-PC contents and structure in the nursing curriculum were conducted in the course syllabus. To collect data related to the education program, the researchers accessed the HAIs-PC module in the curriculum which is published on the website of the institution.\n\nThe qualitative instruments were used to guide the interview of managers and group discussion of teachers and clinical tutors. The University Board interview guide explored the perceptions of the participants from each institution, on the HAIs-PC program in their nursing curriculum with guiding points of (1) educational opportunities, (2) HAIs-PC contents in the nursing course, (3) the duration of the module, (4) used active teaching and learning methods, (5) institutional actions for the improvement, (6) students’ learning opportunities, and (7) students’ learning outcomes. The lecturer focus group topic guide explored the perception of the nursing faculties for (1) the HAIs-PC contents; (2) the study plan, (3) the continuity between the curricular units, (4) the concept of students’ entrepreneurial skills, (4) institution effort for students and teachers’ competencies, (5) equipment, technologies, or teaching materials, (6) effective learning methods, (7) theoretical references, (8) the teaching process. The tutor focus group topic guide explored the perception of the participants on the HAIs-PC program with guiding points of (1) awareness of the nursing students on HAIs-PC, (2) institution support, (3) active learning methods, knowledge gap, (4) students’ challenges and solutions, (5) students’ learning outcomes and (6) hospital and university relationship. The interview and focus group guides were developed by the English-speaking researcher for use in several countries and then collaboratively refined and professionally translated for cross-language use in the local Vietnamese and Cambodian context and culture.\n\nThe translated instruments were validated by five lecturers who met the same criteria as the prospective participants. The lecturers were asked to evaluate individual items on the instruments as well as the entire instruments. The primary investigator and a research assistant then revised the contents of the instruments following the lecturers’ comments and suggestions. The two research assistants in each HEI were nursing teachers who taught nursing students related to IPC. They were trained to collect data using the four research instruments by the primary investigator.\n\nAll the above instruments can be found in the Extended data.\n\nPreceding the data collection, the study was approved by the Health Sciences Research Unit: Nursing (UICISA: E) of the Nursing School of Coimbra (Portugal) with the reference number P761-3/2021; Ethic Committee of Biomedical Research at the Nam Dinh University of Nursing with the approval number 831/GCN-HĐĐĐ (April 22, 2021); Ethical Review Board for Biomedical Research, Hai Duong Medical Technical University with the approval number 191/QD-DHKTYTHD (March 26, 2021); Ethics Committee at the Bolyno Institute (April 22, 2021); and IU Ethics Committee at the International University (April 22, 2021). The acceptance for data collection was obtained from the Asian partner universities. Study information was provided in Vietnamese and Cambodian, and participants were encouraged to ask questions before they signed the informed consent. The quantitative data collection and the face-to-face interviews and discussion groups were conducted in April, 2021 at the campus or at the hospital premises. The participants were informed about the study and its objectives, as well as the voluntary nature of participation. Alphanumeric characters were used instead of participant’s names in the analysis and publication.\n\nIn order to collect the indicators related to the HAIs-PC education program, the research team used data available from the curriculum information at the universities. Moreover, the perception of HAIs-PC education was collected through in-depth interviews and focus group discussions. At the beginning of the focus group session and interviews, explanations about the research goals and regulations were given to the participants. The interviews and group discussions took about 30 to 60 minutes in duration. All interviews and group discussions were recorded via tape recorders. Immediately after each interview or group discussion, the data analysis was conducted to ensure that data saturation was reached. The location and time of the interviews were selected based on the participants’ preferences. After holding interviews, in case of any further questions, the researcher contacted the participants by referring to them by phone calls or e-mail concurrent to data collection.\n\nSee Extended data: University Board’s interview and group discussion guide.\n\nThe interview recordings were transcribed by the researchers. The content analysis with the inductive method was used for data analysis.17 The researchers conducted a separate preliminary analysis of interview and group discussion transcripts. The data were read line by line, and key sentences and phrases were allocated, underlined, and coded. Similar codes were combined, and primary themes were made. Data reduction was conducted in all analyzed units until categories emerged, so that general, conceptive, and inductive data were put in the main theme. The researcher held meetings to edit and recode the themes in the process of data analysis. The meetings provided an opportunity to delineate, define themes, and discuss cultural and linguistic perspectives. The saturation of the data was reached after eleven interviews and six group discussions, but scheduled interviews and group discussions were completed and provided support for previous themes. The researchers continued the interactive process until an agreement was reached on the themes. The data of training records were analyzed simultaneously with the content analysis.\n\n\nResults\n\nThe participants were asked questions pertaining to the focus of the implementation of HAIs-PC education, the challenges of the faculty on teaching and students’ learning, facilities, and students' competency perceptions. The results indicated that there were challenges in HAIs-PC education in Vietnamese and Cambodian institutions. The challenges come from the curriculum, students’ and teachers’ characteristics, and universities. The diagnostic indicator results provided primary information on the designed curriculum regarding each academic year for the HAIs-PC module, contents, and credit hours for theory and practice. The interview and group discussion results showed that the challenges came from the implementation of the curriculum, an inactive learning environment, and unsupportive facilities at the university.\n\nMost HEIs have been providing nursing education programs for bachelor students for more than ten years. These universities integrated HAIs-PC contents into the nursing curriculum during various study years. Mostly, nursing teachers provided lectures and lab practice of HAIs-PC to the nursing students in the second year of the study program. The students practiced providing care for patients in the hospitals in the third year of study, under the supervision of the clinical lecturers and nurses.\n\nThe total participants in the study were 49 managers, nursing faculty teachers, and clinical tutors. Vietnam (V) had 28 participants, with university 1 (U1) (16 participants), University 2 (U2) (12 participants), and Cambodia (C) had 21 participants, with university 3 (U3) (12 participants) and University 4 (U4) (9 participants). The detail is shown in Table 1.\n\nThe diagnostic indicators of the curriculum\n\nThe four universities offered a wide range of nursing degree levels. Universities in Vietnam had bachelor's degrees in nursing with large numbers of students, especially U1 currently has 1,733 nursing bachelor's students studying at the university. All universities in the countries lectured one to two credits for the HAIs-PC module, which, distributed time equally between theory and skill practice. In Asian countries, each credit corresponds to 15 hours of theory or 30 hours of skill practice. The institutions provided lecture and skill practice hours for HAIs-PC variously, U1 with 45 hours and U4 with 15 hours for the whole course. Clinical practice at the hospital for HAIs-PC was integrated into the other modules. All four universities provided students with knowledge and lab practice related to HAIs-PC. The contents were transmission routes and HAIs prevention measures; sterilization for and management of medical equipment; Sanitization of the hospital environment; Medical waste management; Prevention and control of blood and body fluid exposure and Infection surveillance. There was a slight difference in the program structure. A university in Vietnam arranged an HAIs-PC course with 3 credits, including a credit in microbiology and parasites HAIs-PC theory and clinical practice. The other universities provided the program with a different structure. The total hours of theory and practice ranged from 15 to 45 hours, in which, the practice hours were double compared to theory hours. Details are presented in Table 2.\n\nResults of current challenges of HAIs-PC education in the nursing curriculum in Asian countries\n\nThe research findings were structured into three generic themes, which were the Implementation of HAIs-PC education into nursing curriculum, Positive aspects fostering IPC learning, and Negative aspects hindering HAIs-PC learning (Figure 1).\n\nImplementation of HAIs-PC education into the nursing curriculum\n\nThe generic theme was composed of sub-themes including Curricula’s structure and content and Used teaching methods. The HAIs-PC education in the four institutions was recognized as an important aspect of nursing education. However, the Curricula’s structure and content in the institutions were different depending on the perception of the role of HAIs-PC in clinical settings. The participants indicated various perceptions regarding the current nursing curriculum in their universities. In both countries, the nursing curriculum contents, teaching materials, and teaching methods met the objectives related to the HAIs-PC course. The HEI regularly updated the structure and contents of HAIs-PC contents to meet the rapidly changing situation of the countries’ healthcare system and the needs for healthcare services. The participants indicated that continuous updating of the contents of the course always ensured a scientific and evidence-based curriculum before integrated teaching to students. Moreover, it seemed that the students gained the needed basic knowledge and skills on HAIs-PC during their learning process.\n\n“Earlier the HAIs-PC unit focused only on the contents related to HAIs-PC, but now the unit is integrated with Microbiology and Parasitology. Therefore, this provides for students with an overview of the knowledge related to infection control” (VG2)\n\nA difficulty in the implementation of HAIs-PC education in the curricula was that the countries did not have clear enough guidelines from the Ministry of Health (MOH) and there was no common agreement between the academic and the hospital settings on HAIs-PC curriculum. Therefore, the Universities were not able to prioritize the corresponding HAIs-PC practical skills before students practiced in the hospital settings.\n\n“We would like to have a specific guideline from the MOH regarding HAIs-PC training curricula that has consistency regarding the integration of knowledge and practice with clinical practice, and between the academic and clinical settings.” (CG2)\n\nHowever, the Covid-19 pandemic brought more consistency into the regular updating of teaching content met the need of healthcare service systems under the direction of MOH and WHO.\n\n“Especially with the outbreak of the Covid-19 pandemic, we had updated the contents related to the HAIs-PC such as the use of personal protective clothing, cleaning, and disinfection” (VG1)\n\n“The lecturers participated and received certificates of open courses on HAIs-PC regarding Covid 19 by WHO at the request of the University to update knowledge for all students”\n\nThe used teaching methods were based on an assumption that nursing bachelor’s students need to gain enough knowledge and skills related to HAIs-PC while they study in classes and skill practice before they enter the clinical settings. Therefore, the universities designed a curriculum that provided bachelor students the lectures on basic knowledge and skill practice, in which the strategies and methods in teaching HAIs-PC were used to meet the requirements.\n\n“Simulation method or teaching based on clinical situations is very important and necessary, it helps students connect knowledge and skills together as well as help students get closer to clinical reality”\n\nThe lecturers have precepted the role of simulation in clinical teaching, especially in training students in positive thinking and decision-making skills for HAIs-PC. However, their capacity was insufficient to apply the method. In clinical settings, awareness of the exited inherent gap between the academic and clinical settings, the wards tried to create the best possible conditions to guide students while practicing HAIs-PC in the hospital.\n\n“Clinical experience and practice are very important to build simulation situations; some young teachers have difficulty implementing this teaching method” (VG1&2)\n\n“…., new teaching methods need to be trained, … we have not been properly trained in simulation teaching methods … so it is difficult to apply” (CG1)\n\nThe research results indicated that the Positive aspects fostering HAIs-PC learning were composed of the sub-themes of Students’ motivation to learn and the Possibilities to practice clinical skills. Most of the universities informed that they tried to motivate students’ HAIs-PC learning by using different and more interactive teaching methods that promoted greater students’ participation. They also emphasized the need to combine different teaching methods to promote the integration of theory into practice. Moreover, dealing with the lack of practical equipment and tools, the University arranged extra-curricular laboratory rooms for students so that they had more opportunities to practice HAIs-PC skills.\n\n“We arrange instructor and senior students to support other students to practice on HAIs-PC skills at extra-curricular practice rooms” (CI1)\n\nStudents were encouraged to participate in hospital quality improvement activities and research projects related to HAIs-PC in order to increase their interest in learning and enhance the student's experience in the module. Students, who conducted research or participated in data collection for a project, were awarded points for completing the study program at the University. Some universities considered that quality improvement activities and research projects on HAIs-PC should be emphasized in order to enhance the lecturers’ quality and encourage student participation.\n\n“Many students participate in HAIs-PC research and quality improvement activities regarding HAIs-PC, students showed their innovative, creative thinking, this learning activity is quite helpful for students”\n\nThe HEIs enhanced the possibilities to practice clinical skills related to HAIs-PC among students. The nursing curriculum in the countries does not include skill practice of HAIs-PC in clinical settings, which is integrated with other clinical modules. However, in recent years, a number of young lecturers were trained to use simulation pedagogy. Therefore, the Universities designed practical teaching using simulation pedagogy, with several of modern HAIs-PC equipment to increase students' practice opportunities.\n\n“Need to build a standard simulation to practice HAIs-PC. However, the standard simulation does not mean that we need to build a simulation in the nursing skill lab; we can integrate simulation into our hospital in which our students practice infection prevention and control” (VG1)\n\nThe generic theme “negative aspects hindering HAIs-PC learning” was composed of the sub-themes Lack of hospital and university cooperation, Lack of learning materials and equipment, and Teachers’ inappropriate teaching methods of HAIs-PC. There was uncertainty among the leader and teacher groups about how HAIs-PC education was provided and if it was their responsibility. Many interviewed expressed uncertainties expressed as Lack of hospital and university cooperation. The managers indicated that the institutions had regular meetings with hospital leaders for implementing clinical training in the hospitals. Moreover, most of the institutions focused on enhancing the quality of teaching HAIs-PC in clinical settings. However, there was a number of factors affecting the quality of students' clinical skills training that they considered beyond their capabilities, e.g. challenges for learning when there were too many patients and students in the clinics.\n\n“There are many students taking clinical placements at the same time in one ward leading to difficulty for tutors and lecturers to manage students in implementing HAIs-PC” (CG2)\n\nSome of the visiting lecturers, who worked at hospitals, stated having lack of pedagogical skills and theoretical knowledge of HAIs-PC education. Even though the universities had a training plan, the lecturers´ could not always attend the courses because of work overload at the hospital.\n\n“Lack of uniformity in teaching method, and assessment method between tutors lead to students experiencing different wards; they receive different guidelines and assessment about HAIs-PC. These results in difficulty in identifying student weakness to assist students” (VG1&2)\n\nThe results of the group discussion and interview indicated that the universities were lack of learning materials and equipment for HAIs-PC education. Hospital settings were very important in maintaining and developing students' HAIs-PC skills because students gradually improved their HAIs-PC competency during a period of clinical practice. The university always tried to invest in equipment. The lecturers taught practical skills by imagining the equipment because the equipment and infrastructure did not meet the requirement for practice. Many contents related to the practice of HAIs-PC were not taught in the skill labs. Therefore, the students could only observe these skills in the hospital such as clinical waste management, management of transmission routes, and sources of transmission.\n\n“Lack of equipment for teaching and practice of infection control is a problem … It is difficult to be able to equip a full set of equipment for HAIs-PC in academic settings because it is very costly. University should cooperate with the hospitals where HAIs-PC equipment is satisfactory so that students can experience”\n\nThe university efforted to increase learning resources for students such as updating the textbooks every two years and increasing the books in the library. However, these efforts also failed to meet the current training needs of HEI, as quoted:\n\n“Some of the textbook contents were outdated … Most students only use the required textbooks, rarely learn more from other resources …encounter problems with database access and language barriers” (VGs&CI1). “There is a lack of teaching tools for practice such as nursing procedure manuals or videos to assist students in self-studying” (VG2)\n\nMost of leader Boards and teachers indicated that the teachers currently used inappropriate teaching methods of HAIs-PC. The need to use different and more interactive teaching methods that promote greater participation with more active methodologies was highlighted. Teachers also emphasized the need to combine different teaching methods to enhance the competency of students in clinical settings. However, the application of these methods encountered difficulties. The capacity building of lecturers should have been focused on developing active learning and teaching skills such as case- and problem-based education. These methods could have helped students connect knowledge and skills together as well as help students get closer to clinical reality. Currently, in the HEI, there was a gap between theory and practice in HAIs-PC education, as students did not have a chance to practice with the functioning equipment.\n\n“Lecturers mainly provided students with purely theoretical knowledge…while practicing, students performed procedures using the checklist with few real clinical situations or simulation scenarios … students shown less critical thinking, problem-solving and teamwork skills after completing the course” (CGs & VG1)\n\nThe students performed well on the basic skills of HAIs-PC in the skill lab, however, while taking care of real patients in the clinical settings, students sometimes became confused. The students had difficulty in making decisions and following technical procedures as well as the principles of HAIs-PC correctly. The reason was that students were not familiar with clinical situations before practicing in the hospitals but were just taught single practical skills. The positive learning skills, such as critical thinking, problem-solving, and teamwork, were emphasized only in theory classes instead of skill practice. Therefore, simulations may be a good teaching method in the future in Asian countries, because it helps to combine a variety of cognitive, psychomotor, and emotional skills.\n\n“When asking students to do single skills such as hand hygiene or personal protective equipment, students do the right thing and are quite proficient but when put in a specific situation and patients, students sometimes forget to perform it or performed incorrectly” (CG2)\n\nAlthough, the contents and practice of HAIs-PC were integrated into clinical-related modules. The clinical teachers and tutors were not confident in their integrated knowledge of HAIs-PC, then they rarely strengthen the HAIs-PC practice in clinical settings.\n\n“Having many nursing subjects applying the principles of HAIs-PC in the delivery of nursing care, for example, fundamental of nursing, medical-surgical nursing clinical practice, however, the instructors rarely further reinforced IPC skills while practicing in hospitals” (CG1)\n\nThe students lacked the initiative to apply HAIs-PC skills or avoided or missed out on safety awareness in providing care to the patients in clinical settings. The leaders attributed the cause to the traditional culture of providing a passive learning environment for students. Moreover, the large number of students in theory and practice classes weakened lecturers´ possibilities to reach all the learning outcomes. Furthermore, lecturers should be retrained periodically to update their HAIs-PC expertise to become more confident in the teaching methods. Updating this knowledge also helps lecturers at institutions have uniformity in teaching methods.\n\n“Many nursing teachers do not feel confident in teaching the theory of infection and prevention subject because they have not regularly received training on infection and prevention subject” (VG2)\n\n“We made great efforts to build an e-learning course on HAIs-PC to promote autonomy, self-study, and problem-solving for learners. The relevance and applicability of the learned knowledge about IPC at the University in the clinical settings was a common concern” (VI2)\n\n“A practice group can be up to 20 to 25 students; this is very difficult to manage whether students achieve other learning objectives set out in the lesson or not” (VG2)\n\n\nDiscussion\n\nThis study explored the challenges of HAIs-PC education in nursing curriculum in two Vietnamese and two Cambodian HEIs. The exploratory study was conducted in two Vietnam and two Cambodia HEIs with 49 participants who were leaders, nursing teachers, and clinical tutors. There was diversity in the ratio of students to faculty between universities and between countries, specifically, the ratio in a Vietnam university was 22.7 while that at Cambodia university was 12.0. The research results indicated that there were various hours in theory and laboratory practice from university to university. Furthermore, there were differences in the hours per credit requirement between the two countries, particularly with regard to the number of hours per credit for practical courses. The results correspond to the results of previous studies on the diversity in the regulation system of nursing professional training of two countries.18,19 The qualitative results emerged the main theme challenges in HAIs-PC education with generic themes of Implementation of HAIs-PC education into nursing curriculum, Positive aspects fostering HAIs-PC learning, and Negative aspects hindering HAIs-PC learning.\n\nResearch results on the positive aspects of HAIs-PC education at the HEIs included motivating students’ learning and enhancing students' ability to practice. HEIs strived to apply active teaching methods to enhance student engagement in learning. The addition of an extra-curricular skill practice for students was also a positive method to be applied. In addition, the case-based pedagogy had begun to improve students' practical capacity on HAIs-PC. The research results were similar to previous studies to enhance the learning and practice of HAIs-PC of nursing students.20,21 Kim and colleagues (2020) indicated that simulation pedagogy improved HAIs-PC competence by increasing knowledge, compliance, and practical quality and outcomes in patients.22\n\nThe negative aspects hindering HAIs-PC learning were revealed by the managers, lecturers, and clinical tutors. The participants admitted that they used active teaching methods in delivering HAIs-PC lectures, but it was not effective. These perceptions were consistent with nursing instructors in some Asian countries because they were familiar with traditional teaching methods, along with a large number of students in study groups.23,24 The connection, application of knowledge, and practice between the University and the clinical environment was also a challenge to the teaching capacity of lecturers. Therefore, improvements are urgently needed in HAIs-PC education for nursing bachelors at Asian universities. To create a positive learning environment, specifically related to HAIs-PC education, efforts need to come from teachers, students, and especially from educational institutions. Therefore, in order to improve the challenges in HAIs-PC education for nursing students, HEIs of Vietnam and Cambodia should pay attention to the following changes and improvements. Firstly, the universities should build an active learning model in which increased content knowledge, critical thinking, problem-solving abilities, and positive attitudes toward learning and make a fluency between theory and clinical practice.25 Although leaders and teachers claimed that they regularly updated the books, however, updating should focus on making the books more appealing to learners. Instead of simply providing content, the books should include images and interesting real-life cases related to HAIs-PC. This may make an interesting first impression of HAIs-PC learning. Moreover, the orientation session is equally important to help students understand the significance of HAIs-PC learning in their professional practice. In theory, the teachers should use active teaching methods, create two-way interaction and initiative in learning, and provide timely two-way feedback. For practice, the simulation pedagogy would provide students with the opportunity to familiarize themselves with and experience HAIs-PC before practicing in real clinical settings.26\n\nUnsupported facilities for HAIs-PC learning included the lack of equipment, teaching materials, and clinical facilities. This result corresponds to some previous studies.27,28 Through the nursing education system, lecturers and instructors should be empowered and encouraged to provide an enhanced nursing education approach that applies local facilities and resources, as well as enhance awareness of local resources. Developing countries’ universities should adopt and adapt active teaching methods to improve nursing education by utilizing available resources.29,30 The use of different active learning methods, especially, simulation pedagogy may improve communication and critical thinking skills and enhance safety in healthcare for the patients and nursing students themselves.31\n\nThe research results indicated that one of the challenges was the lack of knowledgeable and skilful lecturers in the field of HAIs-PC education. Many nursing teachers were not confident in teaching theory and practice on HAIs-PC because they have not been regularly trained on the module. The results were congruent with previous studies that the teachers need to be more competent by attending regular training or workshops on HAIs-PC education.32 The InovSafeCare Pedagogical Model for HAIs-PC education was implemented and presented the effectiveness of student HAIs-PC outcomes in Europe.33 The model focuses on the comprehensive application of active teaching pedagogies to theoretical, practical, and clinical learning environments. In particular, the simulation method with well-designed scenarios based on clinical reality enhanced students’ ability to critically thinking and problem-solving HAIs-PC problems.34 A similar culturally adapted model should be piloted to evaluate the model on HAIs-PC students’ outcomes. Moreover, the universities should have regular training on HAIs-PC for the teachers, as well as assign them as clinical instructors in the HAIs-PC department at the hospital for their updating knowledge and skills. Besides supplementing competency related to HAIs-PC, lecturers also need to be trained to improve their pedagogical skills as well as how to deploy active teaching methods, especially simulation teaching. In addition, the capacity building of evidence-based research and practice in the field of HAIs-PC should also be considered to enhance students’ and lecturers’ competency in HAIs-PC.35,26\n\nThe present study used a qualitative design with in-depth interviews and focus group discussions on diverse participants and a quantitative design to ensure various perspectives were obtained from the participants. In addition, the number of focus groups and in-depth interviews was continuously conducted until the final interview and discussion, although the researcher found the data were saturated, which made the research results identified rich and reliable. However, only data from leader boards, teachers, and tutors were collected, without perceptions from students. Future research should be explored these challenges from the student’s perspective for a holistic perspective on the issue.\n\nThe research results identified that challenges in HAIs-PC education were considered sensitive topics for their HEIs. However, face-to-face interviews and focus group discussions with participants may create the impression that participants could be trying to “hide” real challenges or overemphasize the positive aspects of their HEIs. During the interviews and focus group discussions, the researcher emphasized that the research purpose was to identify common challenges, not problems with the participants themselves, therefore, reducing concealment. In addition, interviews and focus group discussions conducted in universities had various characteristics and cultures, the research results may be generalized to the other HEIs in Asian countries.\n\n\nConclusions\n\nThe study may provide evidence of the challenges to the current HAIs-PC education in the nursing curriculum in Vietnam and Cambodia. The negative aspects hindering HAIs-PC education of inappropriate pedagogy, equipment and materials, and university and hospital cooperation were the challenges. The evidence may suggest useful solutions for future nursing education on HAIs-PC practice, consequently reducing the prevalence of HAIs in Vietnam and Cambodia.\n\nProviding a robust, updated HAIs-PC education curriculum that is culturally consistent and contextually relevant is an important first step in improving nursing education outcomes and quality care. The faculty and clinical instructors, who provide direct education to nursing students, are ideally positioned to develop students' HAIs-PC skills for future nurses with safe and effective care competency. Regularly updating content, applying active teaching methods in skills training, and utilizing existing local resources may improve the effectiveness of HAIs-PC education for nursing students.\n\n\nDisclaimer\n\nThe European Commission's support for the production of this publication does not constitute an endorsement of the contents, which reflect the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.", "appendix": "Data availability\n\nFigshare: Current challenges in teaching HAIs-PC in nursing education in Vietnam and Cambodia: a qualitative study. https://doi.org/10.6084/m9.figshare.23669277.v2. 36\n\nThis project contains the following underlying data:\n\n- HAIs-PC data\n\nThe qualitative data underlying this study are available on request from the corresponding author. The data are not publicly available due to ethical considerations regarding personal information and to respect what was written in the signed informed consent.\n\nFigshare: Current challenges in teaching HAIs-PC in nursing education in Vietnam and Cambodia: a qualitative study. https://doi.org/10.6084/m9.figshare.23669277.v2. 36\n\nThis project contains the following extended data:\n\n• 26-items record collection instrument\n\n• The University Board’s interview guide\n\n• The lecturer focus group topic guide\n\n• The tutor focus group topic guide\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nThe authors of this study would like to acknowledge the support provided by QUVAE Research and Publications. They were instrumental in guiding the deposition of the underlying and extended data to the Figshare repository.\n\n\nReferences\n\nBoev C, Kiss E: Hospital-acquired infections: current trends and prevention. Crit. Care Clin. 2017; 29(1): 51–65. PubMed Abstract \| Publisher Full Text\n\nWHO: Healthcare-associated infections. Fact Sheet. (Accessed 1 August 2021). Reference Source\n\nMaki G, Zervos M: Health Care-Acquired Infections in Low- and Middle-Income Countries and the Role of Infection Prevention and Control. Infect. Dis. Clin. N. Am. 2021; 35(3): 827–839. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoi LL, Anucha A, Gilbert M: The burden of healthcare-associated infections in southeast asia: a systematic literature review and meta-analysis. Clin. Infect. Dis. 2015; 60(11): 1690–1699. Publisher Full Text\n\nAlemu AY, Endalamaw A, Bayih WA: The burden of healthcare-associated infection in Ethiopia: a systematic review and meta-analysis. Trop Med. Health. 2020; 48–77.\n\nNgo TML, Nguyen TL, Nguyen TKD: Survey of nosocomial infections at Tien Giang General Hospital in 2018 and related factors.2018. Reference Source\n\nPhu VD, et al.: Burden of Hospital Acquired Infections and Antimicrobial Use in Vietnamese Adult Intensive Care Units. PLoS One. 2016; 11(1): e0147544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe T, et al.: Strengthening Adherence to a Central-Line–Associated Bloodstream Infection Prevention Bundle in a Surgical ICU in Vietnam. Infect. Control Hosp. Epidemiol. 2020; 41(S1): s392–s392. Publisher Full Text\n\nTran DM, et al.: High prevalence of colonisation with carbapenem-resistant Enterobacteriaceae among patients admitted to Vietnamese hospitals: Risk factors and burden of disease. J. Infect. 2019; 79(2): 115–122. Publisher Full Text\n\nHearn P, Miliya T, Seng S, et al.: Prospective surveillance of healthcare associated infections in a Cambodian pediatric hospital, antimicrobial resistance and infection control. Antimicrob Resist Infect Control. 2017; 6: 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaque M, Sartelli M, McKimm J, et al.: Health care-associated infections - an overview. Infection and drug resistance. 2018; 11: 2321–2333. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaleem Z, Godman B, Hassali MA, et al.: Point prevalence surveys of health-care-associated infections: a systematic review. Pathogens and global health. 2019; 113(4): 191–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Global Action Plan on Antimicrobial Resistance. (Accessed 1 August 2021). Reference Source\n\nVietnam Ministry of Health: Circular No. 16/2018/TT-BYT dated July 20, 2018 of the Minister of Health on regulations on infection control in medical examination and treatment establishments, editor.2018.\n\nCDC: Guidelines for Environmental Infection Control in Health-Care Facilities.2019. Reference Source\n\nDarawad MW, Al-Hussami M: Jordanian nursing students' knowledge of, attitudes towards, and compliance with infection control precautions. Nurse Educ. Today. 2013; 33(6): 580–583. Publisher Full Text\n\nElo S, Kyngäs H: The qualitative content analysis process. J. Adv. Nurs. 2008; 62(1): 107–115. Publisher Full Text\n\nChen S: Education and transition to work: evidence from Vietnam, Cambodia and Nepal. Int. J. Educ. Dev. 2018; 61: 92–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujita N, Matsuoka S, Koto-Shimada K, et al.: Regulation of nursing professionals in Cambodia and Vietnam: a review of the evolution and key influences. Hum. Resour. Health. 2019; 17(1): 48. Publisher Full Text\n\nHandiyani, et al.: The effective needle stick injury prevention strategies for nursing students in the clinical settings: a literature review. Enferm. Clin. 2018; 28(1): 167–171. Publisher Full Text\n\nDieckmann P, Torgeirsen K, Qvindesland SA, et al.: The use of simulation to prepare and improve responses to infectious disease outbreaks like COVID-19: practical tips and resources from Norway, Denmark, and the UK. Advances in simulation (London, England). 2020; 5: 3. Publisher Full Text\n\nKim E, Kim SS, Kim S: Effects of Infection Control Education for Nursing Students Using Standardized Patients vs. Peer Role-Play. Int. J. Environ. Res. Public Health. 2020; 18(1): 107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoltzfus JR, Libarkin J: Does the Room Matter? Active Learning in Traditional and Enhanced Lecture Spaces. CBE Life Sci. Educ. 2016; 15(4): ar68. Publisher Full Text\n\nAbdel Meguid E, Collins M: Students' perceptions of lecturing approaches: traditional versus interactive teaching, Advances in medical education and practice. Adv Med Educ Pract. 2017; 8: 229–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBucklin BA, Asdigian NL, Hawkins JL, et al.: Making it stick: use of active learning strategies in continuing medical education. BMC Med. Educ. 2021; 21(1): 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTartari E, Fankhauser C, Peters A, et al.: Scenario-based simulation training for the WHO hand hygiene self-assessment framework. Antimicrob. Resist. Infect. Control. 2019; 8: 58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarzi S, Shahriari M, Farzi S: Exploring the challenges of clinical education in nursing and strategies to improve it: A qualitative study. J. Educ. Health Promot. 2018; 7: 115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Wyngaarden A, Leech R, Coetzee I: Challenges nurse educators experience with development of student nurses' clinical reasoning skills. Nurse Educ. Pract. 2019; 40: 102623. Publisher Full Text\n\nAzad A, Min JG, Syed S, et al.: Continued nursing education in low-income and middle-income countries: a narrative synthesis. BMJ Glob. Health. 2020; 5(2): e001981. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYun B, Su Q, Cai YT, et al.: The effectiveness of different teaching methods on medical or nursing students: Protocol for a systematic review and network meta-analysis. Medicine. 2020; 99(40): e21668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPivač S, Skela-Savič B, Jović D, et al.: Implementation of active learning methods by nurse educators in undergraduate nursing students' programs - a group interview. BMC Nurs. 2021; 20(1): 173. Publisher Full Text\n\nZingg W, Mutters NT, Harbarth S, et al.: Education in infection control: A need for European certification. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2015; 21(12): 1052–1056. Publisher Full Text\n\nPairreira P, et al.: InovSafeCare - Educating students for innovative infection prevention and control practices in healthcare setting, Erasmus Project. ( 2022. Reference Source\n\nKim J, Park JH, Shin S: Effectiveness of simulation-based nursing education depending on fidelity: a meta-analysis. BMC Med. Educ. 2016; 16: 152. Publisher Full Text\n\nHabboush Y, Yarrarapu SNS, Guzman N: Infection Control. [Updated 2022 Jun 5]. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Reference Source\n\nAnh TT & ; PrevInf Group: Current challenges in teaching HAIs-PC in nursing education in Vietnam and Cambodia: a qualitative study. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "276829", "date": "14 May 2024", "name": "Khin Thandar Aung", "expertise": [ "Reviewer Expertise nursing education", "e-learning", "MOOC course", "emergency nursing", "oncology nursing", "public health emergency" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 emphasises the significant challenges in HAIs-PC education in Asian higher education institutions. To improve professional safety and enhance practice outcomes, universities need to address these challenges by implementing suitable teaching methods and ensuring sufficient resources for practical training. Future research should concentrate on evaluating the effectiveness of interventions that aim to overcome these barriers and improve HAIs-PC education in nursing curricula.", "responses": [] }, { "id": "276830", "date": "25 May 2024", "name": "Jaouad El-Hilaly", "expertise": [ "Reviewer Expertise Nursing Education", "Therapeutic education", "Teachers training", "Psychometrics", "Physiology and Pharmacology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 qualitative research, data triangulation through FGs and IDI interviews may lead to a more comprehensive knowledge of the topic of interest. The authors have an acceptable Data source triangulation, they collect data through interviews with individuals (IDI interviews) or groups (FGs). However, some issues need addressing to validate the trustworthiness of findings:\n\nThe number of participants of each FG has been mentioned in Table 1, but there is no information about the participants interviewed individually. The data source of both methods should be reported distinctly. How do both methods (IDIs and FGs) lead to the convergence of the data? to understand how various types of data contribute to understanding of phenomena How does the combined data enhance understanding of the current issue, and answer the research question? Compare the results of both methods of data collection (IDIs and FGs) HAC-PC should be defined in the abstract. Remove the acronym in the title if the submission guidelines do not permit it. Thematic Analysis Software is not reported. What are the limitations of this research 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11944", "date": "02 Jul 2024", "name": "Truong Tuan Anh", "role": "Author Response", "response": "Thanks for the valuable comments. Kindly find the responses to your comments below, 1. The number of participants of each FG has been mentioned in Table 1, but there is no information about the participants interviewed individually. Response: 49 people met the requirements to participate in the study, of which 12 managers participated in individual in-depth interviews. At the same time, 8 group discussions were conducted with 37 lecturers and clinical instructors. 2. The data source of both methods should be reported distinctly. Response: All in-depth interviews and group discussions were anonymous, and each participant was given a code. The interviewers recorded and transcribed data throughout the interviews. The researchers conducted total of 12 in-depth interviews with 12 University Board members and 8 group discussions from lecturers and tutors (4 groups of lecturers and 4 groups of tutors). 3&4. How do both methods (IDIs and FGs) lead to the convergence of the data? to understand how various types of data contribute to the understanding of phenomena. Response: We added a description in the discussion to explain for the importance of various data to contribute to understanding of phenomena. 5. How does the combined data enhance understanding of the current issue, and answer the research question? Response: The present study used a qualitative design with in-depth interviews and focus group discussions on diverse participants and a quantitative design to ensure various perspectives were obtained from the participants. In addition, the number of focus groups and in-depth interviews was continuously conducted until the final interview and discussion, although the researcher found the data were saturated, which made the research results identified rich and reliable. The combined data provided a comprehensive insight into the prevailing challenges of HAIs-PC education in nursing curricula across Asian countries. By analyzing these diverse data sources, researchers could gain a nuanced understanding of the multifaceted issues surrounding HAIs-PC education, including curriculum effectiveness, educational strategies, and barriers to learning. The quantitative data offered statistical evidence regarding the prevalence and impact of HAIs, while deep interviews and focus group discussions facilitated the exploration of nuanced perspectives and experiences of educators and students. This integrated approach not only elucidated the current state of HAIs-PC education but also enabled researchers to address the research question by identifying key areas for improvement and informing evidence-based interventions to enhance IPC learning outcomes in nursing education within Asian contexts. 6. Compare the results of both methods of data collection (IDIs and FGs) Response: The results obtained from both methods of data collection, namely deep interviews conducted with The University Board and focus group discussions with lecturers and tutors regarding their perceptions of the Healthcare-Associated Infections Prevention and Control (HAIs-PC) program in their nursing curriculum, offered complementary insights into the effectiveness and challenges of the program implementation. Deep interviews with The University Board members provided a high-level perspective on the overarching goals, strategic planning, and institutional support for the HAIs-PC program. These interviews may yield insights into the alignment of the program with broader educational objectives and institutional priorities. On the other hand, focus group discussions with lecturers and tutors delved into the frontline experiences, perceptions, and challenges faced in the delivery of the HAIs-PC program. These discussions overall offered a granular understanding of the practical implementation issues, pedagogical strategies, and student engagement within the program. By combining the results of both methods, researchers can triangulate findings, identify converging themes, and gain a comprehensive understanding of the HAIs-PC program's strengths and weaknesses from both strategic and operational perspectives. 7. HAI-PC should be defined in the abstract. Response: Added a definition of HAIs-PC in the abstract 8. Remove the acronym in the title if the submission guidelines do not permit it. Response: Removed the acronym in the title. Title: Current Challenges in Teaching Healthcare-associated Infections Prevention and Control in Nursing Education in Vietnam and Cambodia: A Qualitative Study 9. Thematic Analysis Software is not reported. Response: The content analysis with the inductive method was used for data analysis. 17 The researchers conducted a separate preliminary analysis of interview and group discussion transcripts. The data analysis process was supported by Nvivo 11 software (QSR International) for facilitating the qualitative content analysis. 10. What are the limitations of this research paper? Response: However, the study has some limitations. Firstly, only data from leader boards, teachers, and tutors were collected, without perceptions from students. Future research should be explored these challenges from the student’s perspective for a holistic perspective on the issue. Secondly, the research results identified that challenges in HAIs-PC education were considered sensitive topics for their HEIs. However, face-to-face interviews and focus group discussions with participants may create the impression that participants could be trying to “hide” real challenges or overemphasize the positive aspects of their HEIs. During the interviews and focus group discussions, the researcher emphasized that the research purpose was to identify common challenges, not problems with the participants themselves, therefore, reducing concealment. In addition, interviews and focus group discussions conducted in universities had various characteristics and cultures, the research results may be generalized to the other HEIs in Asian countries." } ] } ]	1	https://f1000research.com/articles/12-1535
https://f1000research.com/articles/13-842/v1	29 Jul 24	{ "type": "Systematic Review", "title": "Effectiveness of Tunnelling Modification Technique Using Platelet-rich Fibrin and Connective Tissue Graft in Gingival Recession: A Systematic Review", "authors": [ "Popy Sandra", "Esti Cahyani Adiati", "Nurul Khairiyah", "Benso Sulijaya", "Yuniarti Soeroso", "Esti Cahyani Adiati", "Nurul Khairiyah", "Benso Sulijaya", "Yuniarti Soeroso" ], "abstract": "Background Gingival recession is a concern in aesthetic and functional perspective. The tunneling technique (TT) is one of the effective root coverage treatments in some gingival recession defects and is associated with favorable outcomes. This paper aims to evaluate the effectiveness of platelet-rich fibrin (PRF) and connective tissue graft (CTG) in gingival recession treatment with TT.\n\nMethods This systematic review used the PRISMA method and electronic bibliographic searches were conducted on seven databases (Google Scholar, Wiley, Pubmed, Sage, Ovid Technologies, Quintessence Publishing, Springer) from December 2018 to January 2023. The search focused on randomized clinical trials (RCTs) that reported TT outcomes in the treatment of Miller class I and II recession with a minimum of six months follow-up.\n\nResults Three out of 399 studies met the inclusion criteria. The three selected studies presented PRF and CTG use in multiple gingival recessions to evaluate tissue condition and clinical parameters before and after the surgical procedure. The clinical parameters evaluated were probing pocket depth (PPD), recession width (RW), width of keratinized gingiva (WKG), and vertical depth of recession (VDR). TT with PRF and TT with CTG is effective in treating gingival recession. PRF is well accepted by patients with a less invasive procedure compared to the CTG procedure. However, TT with CTG showed better results in all parameters at follow-up.\n\nConclusions TT with PRF can be used as an alternative to treat some gingival recession defects. However, TT with CTG produced better clinical results in recession closure.", "keywords": [ "Gingival recessions", "platelet-rich fibrin", "connective tissue graft", "tunneling technique" ], "content": "Introduction\n\nGingival recession is defined as apical migration of the gingival margin at the cemento-enamel junction (CEJ).1 Gingival recession is defined as apical migration of the gingival margin at the cementoenamel junction (CEJ).1 Thin biotype, less width of keratinized tissue, prominence, and root proximity make surgical therapy difficult. Many techniques and biomaterials have been developed to treat gingival recession (GR). Connective tissue graft (CTG) and coronally advanced flap (CAF) techniques are generally considered the most effective and more predictable surgical techniques for root coverage treatment with good long-term stability and are therefore declared the gold standard.1 In some cases, the shortcomings of the CTG techniques is inadequate palate tissue thickness and creates a second surgical site that increases morbidity for the patient. To overcome these various disadvantages, various efforts have been made to find alternative materials for CTG.2\n\nPlatelets play a crucial part in wound healing, thus utilizing platelet concentrate can help speed up the process. Growth factors (GFs) released from platelets are important for wound healing and increase cell angiogenesis, proliferation, and extracellular matrix synthesis.1 Platelets have biologically active proteins, and using autologous platelet concentrates is a promising application in clinical situations that require fast healing, as in the case of tissue regeneration in periodontal plastic surgery.1,3 Concentrated growth factor (CGF) is obtained by centrifuging venous blood in a special centrifuge at varying speeds (2400 to 3000 rpm). In CGF isolation, constantly modified rates can be used when obtaining platelet-rich fibrin (PRF). PRF was first developed in France for use in the field of oral and maxillofacial surgery. PRF is classified as a second-generation platelet concentrate because it is made as a natural concentrate without the addition of anticoagulants.1,3\n\nPlatelet-Rich Fibrin (PRF) comprises a fibrin matrix polymerized in a tetramolecular structure, wherein platelets, leukocytes, cytokines, and circulating stem cells are incorporated.4 PRF is high in leukocytes and immune cytokines, such as IL-1β, IL-4, IL-6, and TNF-α.5 During wound healing, the PRF spontaneously generates a dense fibrin network, allowing for slower breakdown and, as a result, delayed release of growth factors to surrounding tissue.5 The release of growth factors from PRF has been observed to last up to 7 days or longer.5–7\n\nSeveral studies have reported various combinations of the “tunnel” approach with CTG to treat multiple recession cases by avoiding vertical flap incisions while maintaining capillary integrity.1 CTG has high predictability and good aesthetic results. This can be seen from most studies, which have predicted root coverage (RC) ranging from 69% to 97%. In addition, this technique requires a suitable donor location.2,8 This systematic review aimed to evaluate the effectiveness of PRF and CTG in treating GR with the tunneling technique (TT).\n\n\nMethods\n\nThis systematic review uses the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) method (Figure 1), and specific questions are created based on the PICOS principle.9,10 Research questions for the literature search are determined based on four crucial elements, namely population (P), which is patients with treatment of gingival recession, intervention (I) with tunneling technique, comparison (C) between PRF and CTG, results or outcome (O), which are the degree of epithelial attachment (DEA), the width of keratinized gingiva (WKG), and the vertical depth of recession (VDR), and study design(s) randomized clinical trial (with follow-up of at least six months after the procedure). This systematic review’s protocol has been registered with ID number CRD42024556227 in the International Prospective Register of Systematic Reviews, or PROSPERO.\n\nIn healthy adult patients who experience soft tissue recession, “what is the effectiveness of using CTG and PRF with tunneling techniques?\" was the main research question for this systematic review. The following standards were used to determine which studies qualified: RCTs/CCTs study design, follow-up of at least six months after the procedure, literature comparing PRF and CTG and tunneling techniques, studies with full text in English, cases of recession in all areas (anterior/posterior, single/multiple), there is data regarding the degree of epithelial attachment (DEA), the width of keratinized gingiva (WKG), and the vertical depth of recession (VDR) in quantitative form. The focus question for this systematic review was, “In healthy adult patients who experience soft tissue recession, what is the effectiveness of using CTG and PRF with tunneling techniques?”\n\nEligible studies were included with the following criteria: RCTs/CCTs study design, minimum follow-up of 6 months after the procedure, literature comparing PRF and CTG and tunneling techniques, studies with full text in English, cases of recession in all areas (anterior/posterior, single/multiple), there is data regarding the degree of epithelial attachment (DEA), the width of keratinized gingiva (WKG), and the vertical depth of recession (VDR) in quantitative form.\n\nUsing a particular search strategy (Tunnelling Technique AND Platelet Rich Fibrin AND Connective Tissue Graft AND Gingival Recessions), an electronic search was conducted on seven databases (Google Scholar, Wiley, Pubmed, Sage, Ovid Technologies, Quintessence Publishing, Springer). A literature search was done to discover English-language publications from 2018 to January 2023. The entire systematic review approach was carried out by four people, by performed a methodical examination across the databases. After deleting duplicate documents, the authors manually reviewed the abstracts and titles. After that, the authors assessed full-text publications to choose the research that satisfied the inclusion requirements. If consensus was not reached, experienced supervisors were consulted for the final decision.\n\nData were extracted from the accompanying full-text articles by the authors using an Excel Spreadsheet (Microsoft) created specifically for this review. The studies was then assessed for the risk of bias using JBI tools. Data extracted included the author’s name, study design, number of patients and number of implants, patient age, implant location, type of soft tissue defect, soft tissue augmentation technique performed, time of procedure follow-up time, and outcomes. During this process, any discrepancies in the review will go through a discussion process between the authors.\n\n\nResults\n\nThe search on seven electronic databases using the supplied keywords yielded a total of 399 papers. More specifically, 3 studies were acquired from PubMed, 200 from Google Scholar, 147 from Wiley, 8 from Sage, 25 from Ovid Technologies, 15 from Quintessence Publishing, and 1 from Springer. The duplication was eliminated carefully using Excel, obtaining 179 repeated studies. A total of 220 studies passed title and abstract screening, with 142 removed due to noncompliance with the author’s specified inclusion and exclusion criteria. As a result, only fourteen research remain to be analyzed. From 14 studies, there are 11 studies that are not include the required parameters and variable. Only three studies met the inclusion criterias (Tables 1 and 2).\n\n\n\n1. VISTA + PRF\n\n2. VISTA + CTG\n\n\n\n• VISTA combined with CTG or PRF (Miller Class I and Class II recession) => predictable results.\n\n• Significant improvement in clinical parameters from baseline to 6 months in both groups.\n\n• CTG is the gold standard. PRF can be used as an alternative\n\n\n\n1. 10 test group subjects (VISTA with PRF)\n\n2. 10 control groups (VISTA with SCTG).\n\n\n\n• (VISTA with PRF) and (VISTA with SCTG), resulting in varying root coverage.\n\n• VISTA + SCTG = superior to VISTA +PRF in all parameters\n\n• VISTA +PRF and VISTA + SCTG stable results\n\n• Both techniques provide excellent color\n\n• VISTA + PRF can be used if the sole objective is to obtain root coverage, provided there is an adequate apical zone of KT\n\n\n\n1. 20 defect sites (9 patients) treated +PRF (Group I)\n\n2. 20 defect locations (8 patients) + CTG (Group II).\n\n\n\n• Pouch and tunnel procedure + PRF or CTG is effective for RC\n\n• Limitations The results achieved on-site with PRF are comparable to CTG (Gold standard) => better patient acceptability and less invasive approach\n\n• Long-term multicenter randomized controlled clinical trials may be needed to evaluate clinical outcomes for autologous PRF: CTG with pouch and tunnel.\n\nThe risk of bias evaluation for the randomized clinical trial was carried out using the Joanna Briggs Institute (JBI) tools, which contains thirteen domains. The JBI is a reliable and valid tool for assessing the likelihood of bias in studies. Each study’s quality was evaluated independently by two reviewers. The JBI score for each study was computed using the checklist, and the results were presented as percentages. The study with a JBI score of 20-49% was considered high risk of bias, whereas those with 50-79% and 80-100% were moderate and low risk of bias, respectively. Table 3 shows the results of the risk of bias evaluations.\n\n\nDiscussion\n\nGingival recession is defined as abnormal apical displacement of the gingival margin in relation to the cemento-enamel junction (CEJ), exposing the root surface. This causes functional and aesthetic issues, such as dentin hypersensitivity accompanied by open, localized, or widespread root surfaces, and is a component of mucogingival deformity, an anatomical defect caused by a variety of etiological factors.11–13\n\nThe goal of regenerative treatment, in addition to addressing aesthetic concerns, is to ensure the stability of soft tissue attachment. According to the research by Zucchelli et al., long-term maintenance of treatment results can be achieved by focusing on the quality of epithelial attachment and enhancing soft tissue volume.11,12\n\nCTG is considered as the gold standard for treating soft tissue defects.14 Anatomically and histologically, the area around the first premolar contains the most fat and glandular tissue, offering substantial volume when considering tissue quantity. The section of the palate from the second premolar to the second molar has more collagen and less fatty tissue and glands, making it an ideal donor site for grafts in the treatment of gingival recession.15 The limitations of CTG include the need for a second surgical site, the thickness of the palatal mucosa, and the shape of the palate. Consequently, alternatives to the Subepithelial Connective Tissue Graft (SCTG) have been explored, such as the acellular dermal matrix (ADM) and collagen membrane. However, using ADM as a substitute for SCTG increases treatment costs, making it less accessible for some patients. Additionally, the source of these biomaterials (typically from cattle or pigs) may be unacceptable to many patients due to religious restrictions. Therefore, SCTG remains the most recommended option for periodontal plastic surgery procedures.16\n\nCovering multiple root recessions presents a greater challenge in periodontitis treatment.17 Among the surgical techniques described in the literature for treating gingival recession are the tunneling technique (TT) and coronally repositioned flap (CAF), which consider the integrity of the interdental papilla. The SCTG technique has demonstrated a high success rate in patients with multiple recessions.18 The pouch and tunneling technique has been widely used and shown varying degrees of success.17\n\nAs a surgical adjuvant, platelet concentrate (PRF) effectively promotes soft tissue healing, facilitates wound closure, and enhances aesthetic outcomes.19 PRF obtained from the same patients has been shown to contain numerous growth factors and has been successfully used in various surgical procedures.19 Because PRF is not derived from another species, it is cost-effective, easy to prepare, and well-accepted by patients. PRF was obtained following the approach recommended by Choukroun et al. No biochemical blood manipulation is required, and neither bovine thrombin nor anticoagulants are needed prior to surgery. To obtain PRF Figure 2, 20 milliliters of venous blood are drawn from the patient via antecubital vein venipuncture and placed in two sterile 10 ml glass test tubes. These tubes are immediately centrifuged at 3000 rpm for 10 minutes, resulting in the formation of a PRF membrane.17,19,20\n\nSource: Own representation based on Mohan et al, 2019.\n\nThe primary goal of this systematic review is to assess the effectiveness of using the TT technique in combination with CTG and PRF. The use of the pouch and tunnel approach alongside CTG or PRF for treating GR has not yet been extensively compared in the literature.20 An electronic search conducted over the past five years identified only three studies meeting the inclusion criteria, which involved the simultaneous use of TT + CTG and TT + PRF. This highlights the necessity for long-term investigations.20 Research by Chandra et al. comparing the regenerative potential of autologous PRF and autogenous CTG on patient satisfaction in terms of postoperative discomfort, reduced root sensitivity, and improved esthetics.16,20\n\nThis study is the first randomized controlled trial conducted to equate the clinical effectiveness of PRF or CTG with the pouch technique and tunneling for the management of GR.20 Chandra et al. stated that the pouch and tunneling + PRF or pouch and tunneling + CTG procedure was effective for RC.16,20\n\nThe limited results obtained from using TT + CTG make PRF is considered to be comparable to CTG (gold standard). This is supported by the evidence that PRF has better patient acceptance and is a less invasive approach. However, long-term multicenter randomized controlled clinical trials may be needed to evaluate the clinical outcomes of the pouch and tunneling technique + autologous PRF compared with the pouch and tunneling technique + CTG.20 The scant evidence presented contrasts with the study conducted by Abu-Ta’a et al., which assessed and compared the clinical outcomes of advanced-platelet-rich fibrin (A-PRF) and CTG in treating Miller class I/II gingival recession in marginal tissues.21 Tested intergroup analysis showed statistically significant differences in recession parameters between groups at 3 and 6 months, with results that were better for the CTG group.21 This study demonstrated that A-PRF and CTG effectively treated gingival recession defects. However, CTG produces better clinical results in reduced recession height and width.21\n\nPRF cannot function as a direct alternative biomaterial like CTG, which is the gold standard. PRF can be applied in cases with contraindications to CTG harvesting of the palate where the patient and physician wish to reduce morbidity and are willing to accept less than optimal results.1,2 PRF does not require a second donor site, is cost-effective, and prevents graft site complications. However, PRF did not improve root coverage as much as CTG in GR Miller classes I and II. These findings are not enough to reveal the actual clinical effect of TT + PRF on the treatment of GR. More studies are needed to investigate and compare the effectiveness of PRF in terms of root coverage.1\n\nHedge et al. stated that although excellent aesthetic results of CTG have been achieved with root coverage occurring in between 69% and 97% of cases, this technique requires a suitable donor site.2 Within the limitations of this systematic review, it can be concluded that CTG still shows long-term results (12–18 months) were superior to ADM regardless of flap design or surgical technique. When CTG retrieval is not indicated, ADM can be a good alternative in treating gingival recession. Further studies with longer follow-up are needed to determine the long-term stability of xenogeneic and allogeneic soft tissue grafts.10\n\nSubbareddy et al. analyzed the VISTA technique with PRF and VISTA with SCTG and obtained various RC results.17 VISTA applied with SCTG showed superior results to VISTA with PRF in all parameters. VISTA with PRF and VISTA with SCTG both show stable results and provide excellent color results.17 VISTA with PRF can be used if the sole aim is to obtain root coverage provided an adequate Keratinized Tissue (KT) apical zone.17 The studies discussed in this literature state the effectiveness of using TT + CTG and TT + PRF for various dental procedures and periodontal. Jankovic et al. and Aroca et al. found that the combination of TT + CTG and TT + PRF resulted in significant improvements in clinical parameters, such as reduced gingival recession and increased keratinized tissue width. Jankovic et al. stated that using TT + CTG and TT + PRF is an effective approach for soft tissue augmentation and root coverage procedures.10,21 Further research is necessary to assess the long-term stability and success rate of these techniques.22,23\n\n\nConclusions\n\nModern methods, such as CTG and bio-mediators, are anticipated to deliver both aesthetic and functional outcomes in the regeneration of periodontal tissue. The results achieved using both methods show that they are highly successful and can be applied in the treatment of RC. Despite the observed statistical differences, the PRF method is equally suitable as an alternative to CTG for the treatment of RC, given its advantages in patient comfort and simplicity of implementation. The primary statistical difference between the two methods is the amount of keratinized gingiva obtained, which can be related to the histological structure of the soft tissue graft. A wider zone of keratinized gingiva may influence the long-term stability of the results achieved. Although TT + PRF can be used as an alternative to treat some GR defects, TT + CTG produces better clinical results regarding reduced recession height and width. The limitation of this study is that research comparing the use of TT with CTG and TT with PRF is still very limited. PRF does not increase root coverage as much as CTG, so more studies are needed to analyse and compare the effectiveness of TT with CTG and TT with PRF in root coverage\n\n\nEthics and consent\n\nEthical approval and consent were not required", "appendix": "Data availability\n\nFigshare: Checklist for Effectiveness of tunnelling modification technique using platelet-rich fibrin and connective tissue graft in gingival recession: a systematic review, DOI: 10.6084/m9.figshare.26005375.v1. 24\n\nThis project contains the following underlying data:\n\n- PRISMA checklist.docx\n\nFigshare: Figure 1. Prisma Diagram of Effectiveness of tunnelling modification technique using platelet-rich fibrin and connective tissue graft in gingival recession: a systematic review, DOI: 10.6084/m9.figshare.26068684. 25\n\nThis project contains the following und erlying data:\n\n- PRISMA flowchart.docx\n\nData are available under the terms of the Creative Commons Zero “Universal” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nKorkmaz B, Balli U: Clinical evaluation of the treatment of multiple gingival recessions with connective tissue graft or concentrated growth factor using tunnel technique: a randomized controlled clinical trial. Clin. Oral Investig. 2021; 25(11): 6347–6356. Publisher Full Text\n\nHegde S, Madhurkar JG, Kashyap R, et al.: Comparative evaluation of vestibular incision subperiosteal tunnel access with platelet-rich fibrin and connective tissue graft in the management of multiple gingival recession defects: A randomized clinical study. J .Indian Soc. Periodontol. 2021; 25(3): 228–236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohan SP, Jaishangar N, Devy S, et al.: Platelet-Rich Plasma and Platelet-Rich Fibrin in Periodontal Regeneration: A Review. J. Pharm. Bioallied Sci. 2019; 11(Suppl 2): S126–S130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaluja H, Dehane V, Mahindra U: Platelet-Rich fibrin: A second generation platelet concentrate and a new friend of oral and maxillofacial surgeons. Ann. Maxillofac. Surg. 2011; 1(1): 53–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPavlovic V, Ciric M, Jovanovic V, et al.: Platelet-rich fibrin: Basics of biological actions and protocol modifications. Open Med. 2021; 16(1): 446–454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobayashi E, Flückiger L, Fujioka-Kobayashi M, et al.: Comparative release of growth factors from PRP, PRF, and advanced-PRF. Clin. Oral Investig. 2016 Dec; 20(9): 2353–2360. PubMed Abstract \| Publisher Full Text\n\nZwittnig K, Kirnbauer B, Jakse N, et al.: Growth Factor Release within Liquid and Solid PRF. J. Clin. Med. 2022 Aug 29; 11(17): 5070. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReddy S, Prasad MG, Bhowmik N, et al.: Vestibular incision subperiosteal tunnel access (VISTA) with platelet rich fibrin (PRF) and connective tissue graft (CTG) in the management of multiple gingival recession – A case series. Int. J. Appl. Dent. Sci. 2016; 2: 34–37.\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021; 372: n71. Published 2021 Mar 29. Publisher Full Text\n\nKorkmaz B, Balli U: Clinical evaluation of the treatment of multiple gingival recessions with connective tissue graft or concentrated growth factor using tunnel technique: a randomized controlled clinical trial. Clin. Oral Investig. 2021; 25: 6347–6356. Publisher Full Text\n\nFan KA, Zhong JS, Ouyang XY, et al.: Vestibular incision subperiosteal tunnel access with connective tissue graft for the treatment of Miller classI and II gingival recession. Beijing Da Xue Xue Bao. 2019; 51(1): 80–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZucchelli G, Mele M, Stefanini M, et al.: Predetermination of root coverage. J. Periodontol. 2010; 81: 1019–1026. PubMed Abstract \| Publisher Full Text\n\nPazmiño VFC, Rodas MAR, Cáceres CDB, et al.: Clinical Comparison of the Subepithelial Connective Tissue versus Platelet-Rich Fibrin for the Multiple Gingival Recession Coverage on Anterior Teeth Using the Tunneling Technique. Case Rep. Dent. 2017; 2017: 4949710–4949716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKahn S, Araújo ITE, Dias AT, et al.: Histologic and histomorphometric analysis of connective tissue grafts harvested by the parallel incision method: A pilot randomized controlled trial comparing macro-and microsurgical approaches. Quintessence Int. 2021; 52: 772–778. PubMed Abstract \| Publisher Full Text\n\nAzar EL, Rojas MA, Mandalunis P, et al.: Histological evaluation of subepithelial connective tissue grafts harvested by two different techniques: Preliminary study in humans. Acta Odontol. Latinoam. 2019; 32: 10–16. PubMed Abstract\n\nKumar A, Bains VK, Jhingran R, et al.: Patient-centered microsurgical management of gingival recession using coronally advanced flap with either platelet-rich fibrin or connective tissue graft: A comparative analysis. Contemp. Clin. Dent. 2017; 8: 293–304. PubMed Abstract \| Publisher Full Text\n\nSubbareddy BV, Gautami PS, Dwarakanath CD, et al.: Vestibular Incision Subperiosteal Tunnel Access Technique with Platelet-Rich Fibrin Compared to Subepithelial Connective Tissue Graft for the Treatment of Multiple Gingival Recessions: A Randomized Controlled Clinical Trial. Contemp. Clin. Dent. 2020; 11(3): 249–255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZuhr O, Fickl S, Wachtel H, et al.: Covering of gingival recessions with a modified microsurgical tunnel technique: case report. Int. J. Periodontics Restorative Dent. 2007; 27(5): 457–463. PubMed Abstract\n\nKumar A, Bains VK, Jhingran R, et al.: Patient-centered microsurgical management of gingival recession using coronally advanced flap with either platelet-rich fibrin or connective tissue graft: A comparative analysis. Contemp. Clin. Dent. 2017; 8: 293–304. PubMed Abstract \| Publisher Full Text\n\nChandra V, Bains VK, Jhingran R, et al.: Comparative Evaluation of Platelet-Rich Fibrin versus Connective Tissue Grafting in Treatment of Gingival Recession Using Pouch and Tunnel Technique: A Randomized Clinical Study. Contemp. Clin. Dent. 2022; 13(3): 217–226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbu-Ta’a M: Advanced Platelet-Rich Fibrin and Connective Tissue Graft for Treating Marginal Tissue Recessions: A Randomized, Controlled Split-Mouth Study. Cureus. 2023; 15(3): e35761. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJovičić B, Matijević S, Veličković S, et al.: Effectiveness of Plasma-Rich Fibrin and De-Epithelialized Free Gingival Graft in the Treatment of Gingival Recessions. Medicina. 2023; 59(3): 447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSculean A, Aroca S: 12.9 Regenerative Procedures for Root Coverage.\n\nChecklist for Effectiveness of tunnelling modification technique using platelet-rich fibrin and connective tissue graft in gingival recession: a systematic review. DOI: 10.6084/m9.figshare.26005375.v1\n\nPrisma Diagram of Effectiveness of tunnelling modification technique using platelet-rich fibrin and connective tissue graft in gingival recession: a systematic review. DOI: 10.6084/m9.figshare.26068684" }	[ { "id": "313660", "date": "11 Sep 2024", "name": "Triveni M Gowda", "expertise": [ "Reviewer Expertise Inter-relationship of periodontitis with systemic disease", "Laser", "Photo-biomodulation", "Platelet Rich Fibrin", "Osseous defects", "gingival aesthetics", "minimal invasive surgical procedures" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. Overall the article is fairly well written and the topic of research is quite interesting 2. General grammatical errors needs to be corrected. 3. For a review article the number of references taken seem to be low. 4. Overall the major issue that this particular systematic review faces are that the authors have not been able to retrieve 90% of the shortlisted articles. This not only limits the exclusion of quality literature but also limits the scope of systematic reviews only to freely available literature. 5. All the three selected studies have almost a very similar study population as well as sample size. The population on which the studies have been conducted seems to be quite homogeneous and inclusion of only 3 studies that too on the same population for a systematic review does not same to be ideal. 6. Reviewer would suggest the authors to try to retrieve as much as possible of the 64 articles which were not available and include those to do more thorough review of the available literature. ABSTRACT 7. The ‘tunnelling technique’ line is underexplained, and vaguely presented provide appropriate technique. 8. In the background, specify the type of gingival recession. 9. In the result section – “PRF is well accepted by patients with a less invasive procedure compared to the CTG procedure” can be further improved and modified. 10. In the conclusion section – ‘TT with CTG produced better clinical results in recession closure’. Provide a detailed explanation of how it is better with respect to parameters. INTRODUCTION 11. Definition of the gingival recession is repeated. Rectify the same. 12. Mention as ‘root’ prominence in place of prominence. 11. Provide and highlight additional shortcomings of CTG. 13. Specify the isolation rate of CGF. 14. Specify the duration range of PRF in accordance with the most recent articles. METHODS 15. Provide the reference where the ‘degree of epithelial attachment (DEA)’ is considered as an outcome measure for tunnelling procedure in gingival recession procedure. 16. Only 3 reference articles have been provided and VISTA alone has been taken as reference. Justify 17. Correct the spelling of ‘recipient’ in the Figure 2. DISCUSSION 18. Shape of the palate is not a direct limitation. Justify the same. 19. Mention “coronally repositioned flap (CAF)” as coronally advanced flap. 19. How PRF enhances esthetic outcomes. Justify. 20. Long term studies are available for the tunnelling procedures. Add the same in the discussion part. 21. Additional factors aiding for gingival thickness are not mentioned. 22. Why ADM studies are considered for this review /added. Justify?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) No", "responses": [] }, { "id": "320342", "date": "23 Oct 2024", "name": "Shiva Shankar Gummaluri", "expertise": [ "Reviewer Expertise Periodontology", "Implantology", "Platelet Concentrates", "Periodontal Regeneration" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPresent study was a well framed systematic review. Entire structure was made systematically, PRISMA guidelines were followed, search strategies were perfectly executed. Risk of Bias was well explained. Tables were wwll explained. Discussion was well explained and studies were well quoted with updated knowledge. Present systematic review also gave a proper conclusion after sequential analytical steps\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] }, { "id": "320340", "date": "30 Oct 2024", "name": "Mustafa Tunali", "expertise": [ "Reviewer Expertise periodontology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough it is a systematic review written on a current topic, there are important deficiencies: 1. First of all, an attempt was made to compile studies comparing the clinical use of PRF and CTG in the treatment of gingival recession. However, since Tunnelling Modification Technique was also included as a surgical technique requirement, only 3 studies were able to be included in the systematic review. The success of Tunnelling Modification Technique can be evaluated separately with studies comparing different surgical techniques using the same material. This may be a separate systematic review topic. 2. The second important deficiency is that all PRFs are put in the same category without being fully explained. In addition, material thickness, PRF type, and many important factors are not discussed along with surgical technique. This greatly reduces the power of the systematic review.\nIf these important issues are taken into consideration, all these deficiencies are eliminated, and it can be designed to include more than 10 studies, it can be a good systematic review.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-842
https://f1000research.com/articles/13-71/v1	15 Jan 24	{ "type": "Research Article", "title": "Mapping the Nordic Research Landscape for the period 2016-2020: a comprehensive study of research outcomes, collaborations, and impact", "authors": [ "Aparna Narayan", "Bharti Chogtu", "Manthan Janodia", "Raghu Radhakrishnan", "Santhosh K. Venkata", "Aparna Narayan", "Bharti Chogtu", "Manthan Janodia", "Raghu Radhakrishnan" ], "abstract": "Background This article aims to study the research outcomes of five Nordic countries in terms of research publications, spend on R&D, outcomes and collaborations as these are important parameters to understand research thrust of the countries/regions, in addition to their innovation capability.\n\nMethods The research outcomes of the Nordic countries in terms of the total number of publications, coauthored publications, publications with corporate collaborators, citations, the Field Weighted Citation Index (FWCI) and publications in different subject areas were retrieved using Scopus and its associate SciVal. The research outcomes were extracted for five years from 2016-2020. In addition, total population, researcher population and research spend of these countries have been obtained from World Bank data available for the year 2021.\n\nResults The analysis showed that Sweden has the highest population and the highest number of researchers in this region. All countries have the highest number of coauthored publications with the United States, followed by the United Kingdom, except Iceland, which has the second highest number of coauthored publications with Sweden. Denmark, followed by Iceland, stands prominent with reference to having publications with corporate collaborations. Denmark and Sweden have a high percentage of articles in first quartile journals, which is above the average for Nordic countries. Iceland stands at the top with the highest citations, which is depicted by high FWCI. Across subject areas, the Nordic countries have maximum publications in life sciences. Other prominent subject areas include technology and natural sciences.\n\nConclusion On analysing the research landscape of Nordic countries, maximum research output is in the field of life sciences and medicine, and most of the coauthored publications of these countries are with the United States. Denmark, with its exemplary research output, excels with maximum papers in top quartile journals and with maximum corporate collaborations and the highest FWCI.", "keywords": [ "Nordic countries", "Bibliometric", "Research", "Subject profile" ], "content": "Introduction\n\nResearch is one of the metrics used to evaluate the performance of higher education institutions (HEIs). Investments in research and innovations are indications of a country’s development. Nordic countries have been consistently ranked higher for their research capability and innovation competence. Nordic countries, in Northern Europe and the Northern Atlantic, include Sweden, Denmark, Finland, Iceland and Norway. The countries have been among the top 50 Innovative countries according to Global Innovation Index (GII). The Nordic countries are also ranked among the top 50 in terms of Human Capital and Research, and Infrastructure which demonstrates high research capability of these countries.\n\nKautto1 provides a detailed description of the features of Nordic countries in terms of good economy and social performance. The article also outlines the developments during the period from 1990 to 2000. Eskildsen et al.2 presents employee characteristics regarding job satisfaction and intrinsic work motivation in Nordic countries, suggesting that the satisfaction level of employees in Nordic countries is high and that Danish workers are the most satisfied. The political system of Nordic countries and its influence on the functioning of Nordic countries was presented by Lane & Ersson.3 Kangas & Palme4 reports on the expansion of the computer-based economy in Nordic countries and how it affects businesses. Martela et al.5 provides a broad representation of the culture, lifestyle, and other aspects of Nordic countries. This paper points out that all five Nordic countries have been in the top 10 countries listed in the “World Happiness Report.” Bibliometric studies show that Nordic countries have a long tradition of epidemiological research. Furu et al.6 presents data on the drug exposure behavior of the five Nordic countries. Lin & Edvinsson7 presents a detailed analysis of the human capital, market capital, process capital, renewal capital, financial capital, and other indicators of 40 countries from 1994 to 2005. The analysis in this paper identifies Sweden as having the highest overall intellectual capital, followed by Finland, Switzerland, Denmark, the USA, Norway, and Iceland. These results show that all five Nordic countries are among the top 10 for intellectual capital. Along with the regular professional education offered by HEIs, vocational training plays a vital role. Michelsen et al.8 presents a detailed report on vocational training in Nordic countries. Tossebro et al.9 reports on the reforms carried out by countries during the 1990s to improve conditions for people with intellectual disabilities in Nordic countries. The author presents the trends from a political science perspective and in the context of public management. Loof et al.10 presents survey data from the Organization for Economic Cooperation and Development for Nordic countries such as Finland, Norway, and Sweden at the firm level on two key areas of innovation behavior and innovation contribution to economic growth, concentrating on links between productivity and innovation. Glanzel11 surveys publication activity and citation impact in Scandinavian countries from 1980 to 1997 and reports that Nordic countries have strong co-authorship with Western European and North American countries.\n\nClarke et al.12 reports the bibliometric overview of public health research in Europe as a part of a collaborative study under “Strengthening Public Health Research in Europe” using the publications indexed in the Science Citation Index and the Social Science Citation Index for the period from 1995 to 2004. Publication output was analysed in terms of population, gross domestic product, and the burden of disease. This research found that Eastern and Southern European countries have lower numbers than Northern European countries. Melin et al.13 identifies the collaborative pattern of all Nordic countries, the UK, and the Netherlands in work published in science citation indexed journals. The behavior of HEIs in these regions is shown to be more or less similar in the proportion of international and national collaborative publications, with more than 50% of the publications having international coauthors.\n\nGjersvik et al.14 used PubMed to analyse the publication history of Nordic countries such as Sweden, Denmark, Finland, and Norway in dermatology from 1989 to 2008. Articles were analysed based on the country of the first author’s address. Sweden had the highest number of publications, followed by Denmark, Finland, and Norway. Schneider15 presents an analysis of publication details from the NORIA network of Nordic countries, extracting essential information and reporting on the perspectives on reaching excellence. Sandnes16 presents a bibliometric study on human–computer interaction in Nordic-Baltic countries. The results show that Finland, Sweden, and Denmark dominate the region and that collaborative activities are prominent in top-tier conferences compared to entry-level conferences. However, journals are more effective than conferences in generating citations. Features of the International Science Index (ISI) proceedings database, such as information flow, relative attractions, mobility, and country affinity, are analysed by Glanzel et al.,17 along with information on conference locations in the sciences, social sciences, and humanities. Koskinen et al.18 is a bibliometric evaluation of scientific work published in Finland from 1996 to 2005, arising from the use of schizophrenia as a keyword in the Web of Science (WoS) database index. More than 40% of the publications involved international collaboration. Tarkowski19 presents a detailed analysis of publication outcomes in environmental health research across Europe over 10 years, showing that Switzerland produces the highest number of publications in Europe. Rabow20 uses bibliometric data to present a detailed feedback model for the universities and colleges of Nordic countries. Merigo et al.21 analyses research in fuzzy sciences in Nordic countries from a quantitative perspective using the publication index of the WoS database. The analysis provides details of key sections, focusing on journal relevance, authors, institutions, and countries. Hanvold et al.22 presents a systematic review of 12,528 articles published from 1994 to 2014 in major indexing agencies on risk factors for occupational accidents and illnesses among young workers in Nordic countries. Widfeldt23 reports a geopolitical study and its influence on Nordic countries such as Denmark, Finland, Norway, and Sweden. A detailed discussion of urban–rural dimensions in the context of Nordic countries is presented by Knudsen,24 which also reports the economic development of Nordic countries after World War II.\n\nHEIs from Nordic countries (Denmark, Norway, Finland, and Sweden) are studied from 1985 to 2010 by Thomsen et al.25 on parameters such as whether there is a social gap in HEIs and whether privileged groups have been able to maintain advantages in higher education. Finland and Norway display a substantial drop in inequality, Denmark shows a modest drop in inequality, and Sweden shows no signs of inequality decreasing as higher education expands. Airey et al.26 presents the influence of English medium instruction in Nordic countries such as Denmark, Finland, Norway, and Sweden. The authors also present the problems faced by faculties and other stakeholders in this form of instruction. Political dialogue and its influence on the lifestyle of HEIs in Nordic countries are reported by Skogerbo et al.27 The effect of supplementary education on HEIs is reported by Christensen.28 Tikkanen29 presents the effect of the technology-intensive work culture of people in Nordic countries such as Denmark, Finland, Norway, and Sweden. Baccini et al.30 reports quantitative indicators measuring the performance of Italian researchers using their citations from 2000 to 2016, describing self-citation and cross-citation strategies used as indicators in science policy contents. Countrywide bibliometric analysis of publications and average citation impact in oncology for articles published in the WoS are reported by López-Illescas et al.,31 indicating that countries with larger publication numbers showed a decline in average citation numbers.\n\nZacca-González et al.32 reports a bibliometric study of publications in public health, environmental, and occupational health for the period from 1996 to 2011 in Scopus-indexed journals. This research presents a detailed analysis of publications from Latin American quantitative (number of publications), qualitative (citations), and collaborative research. Khiste & Paithankar33 reports a bibliometric study of publications in sciences, social sciences, arts, and humanities published from 2008 to 2016 and indexed in Scopus. Country-level analysis of collaborative publication data using Scopus-indexed data from 2003 to 2016 is reported in Tibaná-Herrera et al.34 Shehatta & Al-Rubaish35 reports the details of publications with country-related self-citation and their impact on total citations and average citations, percentage citations, and productive research from 1996 to 2015 for publications indexed in the Scopus database. Self-citation had a strong impact on a country’s scholarly performance, and the removal of self-citation would show the real impact of a country’s research. A graphical representation of journal and country ranking conducted in SCImago is described by Hassan-Montero36 in a map showing the relational matrix of citation, coauthored citation, and other bibliometrics from Scopus-indexed journals. Archambault et al.37 reports a comparison of the Scopus and WoS databases. Lin et al.38 reports research output on four clusters, namely, countries, research subjects, patterns of research, and publication numbers in WoS, along with the subject-wise clusters across countries. Corrall et al.39 describes bibliometrics and research data management in innovation, network technologies, scholarly communications, and national policy. The identified publications also describe the challenges faced by academic libraries in engaging in data management. Miremadi et al.40 reports a study of innovation and research mechanisms aimed at limiting climate change, presenting a detailed discussion on research on renewable technologies in Nordic countries.\n\nDe la Porte et al.,41 discussed the political reforms in Nordic countries and how those reforms improve the lives of people in Nordic countries. The paper also discusses a survey of various literature related to political reforms in the nation and how they impact research and education. Karseth et al.,42 the authors present various educational reforms/steps proposed by the government to improve the quality of higher education in Nordic countries. Verger43 presents an analysis of how evidence-based policy making and reforms have resulted in an improved higher education landscape and thereby impacted research outcomes. Andersson & Sund44 presents the evaluation of productivity in terms of student achievement in terms of credits, research productivity and staff. Authors provide the details analysis for the above scenarios for the period 2011 to 2016. Sivesind & Karseth,45 presented studying the educational policy of Nordic countries from the viewpoints of researchers from Nordic countries and the US. These publications help to understand the enablers for HEIs to contribute to the research outcome of individuals, institutions and society.\n\nPrevious studies have reported bibliometric analysis in different subject areas and its implications. The Nordic countries rank high for their research capability, which is measured using research publications, research grants and funds received, citations, innovation etc. Studies on the innovation practices of Nordic countries have also been reported earlier. However, this research analyses research outcomes of Nordic countries as a region comparing research outcome of the countries in the region regarding publication, citations, collaborations and focus on different subject areas. The present work elucidates the bibliometrics of Nordic countries (Denmark, Sweden, Norway, Finland, and Iceland). Also a detailed analyses of the quantitative and qualitative aspects of the publications is reported in this paper. Such bibliometric analysis can give an insight to the funding agencies about the thrust research areas in these countries.\n\nThe aim of this study is to compare the research strengths and research outcomes of the five Nordic countries in terms of researcher population, research spending, scholarly outcomes, and international and corporate collaborations. This research also aims to evaluate the scholarly output of each country based on publications in top quartile journals, publications in different subject areas, and the FWCI of each country in different subject areas.\n\n\nMethods\n\nThe data was extracted from SciVal database (www.scival.com) and Scopus databases (www.scopus.com; RRID:SCR_022559) using the search terms “Denmark”, “Sweden”, “Finland”, “Iceland”, and “Norway” under country filter. The data was extracted from 2016 to 2020. The data were extracted on 1 and 2 November 2021. As five years is considered to be an acceptable window for research parameters to be studied, including number of citations that accumulate over a period of time, we selected the previous 5 year window. The data was collected from Scopus/SciVal for the said window as Scopus/SciVal is the largest scholarly and research database. Publication data was examined for scholarly output (number of publications), collaboration percentage (articles published in collaboration with other national and international institutes, and industry), and subject-wise classification number. Citation-related information for 2016 to 2020 also relates to the period from 2016 to 2021. Other data related to population, researcher population, and research spending as a percentage of countries’ gross domestic product (GDP) were obtained from World Bank data (www.data.worldbank.org) on 15 November 2021 for the period 2016-2020. The data was processed using descriptive statistics using MS Excel, which was presented in the form of tables and graphs.\n\nThe countries included in the study were Denmark, Sweden, Finland, Iceland, and Norway. The research outcomes included in the study encompassed publications, collaboration with other countries, academic--corporate collaborations, and the Field Weighted Citation Index (FWCI) of the above countries. Research outcomes from 2016 to 2020 were included in the study. Data from the Faroe Islands and Greenland were excluded. Research outcomes before 2016 and after 2020 were also excluded.\n\nThe data was retrieved from world bank, Scopus and SciVAL as shown in Figure 1. The consolidated data used for analysis of country wide publication outcome is archived in Ref. 46.\n\nA country’s strength in research, innovation, and technological position can be assessed by its human resource strength. The number of researchers per total population is one of the parameters that provides a reasonable idea of the research strength of a country. In the present research, Figure 2 is a comparative plot indicating the population and researcher population of the Nordic countries. Data related to the population and researcher population were derived from the World Bank database (https://data.worldbank.org/data extracted on 15 November 2021) for the period from 2016 to 2020. Data show that the Nordic countries have similar percentages of researcher population, Denmark having the highest, followed by Sweden, Finland, Norway, and Iceland. The researcher population is approximately 0.6% to 0.8% of the overall population across all the Nordic countries, with Denmark having a slightly higher number than the other countries. The researcher population is expected to be a parameter influencing the quality of research. Figure 3 presents a comparison of the researcher population of Nordic countries to the qualitative metric of research in these countries (citation-normalized FWCI). The percentage of GDP spent on research is one of the most important parameters driving research worldwide. The present research supplies a graphical representation of the researcher population vs. percentage of GDP spent on research and FWCI. Sweden spends the highest percentage of GDP on research at 3.5%. Sweden also has the highest researcher population among all Nordic countries. Research spending in other Nordic countries, such as Denmark, Finland, Norway, and Iceland, follows Sweden. The researcher population follows the same pattern. The data show that the researcher population directly correlates with the percentage of research spending on GDP in Nordic countries.\n\nFigure 3 provides information on the relation of the percentage of research spending to GDP to the quality of publication in FWCI terms. Field weighted values are globally normalized citation values for a particular subject. The global FWCI is 1. Figure 3 shows that despite lowest percentage of research spend compared to GDP, the quality of publications from Iceland is the highest, which is followed by Denmark. However, Sweden, Finland, and Norway have similar FWCIs. In general, the percentage spent on research and the researcher population have a direct impact on publication numbers, quality of publications, and citations. In addition, some other parameters impact publication bibliometrics, as discussed in the next sections.\n\nCollaboration is research involving multiple partners contributing to a study. These collaborations may be at the institute level, national level, or international level. Institute-level collaboration involves people from a particular institution working on a common project. National-level collaboration involves people from a particular country joining together to work on a project. International collaboration involves people from different countries. A detailed study was carried out to understand the publication patterns of Nordic countries and their collaborations. Table 1 shows the number of coauthored publications from Denmark with other countries during the 2016–2020 period. The table also indicates the quality of collaboration based on their citation impact on publications. Table 2 provides details of the top collaborative publications from Finland. Similarly, Table 3 shows the outcomes for Iceland. Table 4 shows the output for Norway, and Table 5 shows the output for Sweden. Tables 1 to 5 show that all Nordic countries have the largest number of coauthored publications with the US. The second-largest number of coauthored publications is with the UK. However, Iceland’s second-largest number of coauthored publications is with Sweden. Germany is also one of the most common countries for collaborative research, after the US and the UK. Each of the Nordic countries has different collaborating countries for their fourth collaborative partner. Denmark collaborates with Sweden, the Netherlands, Italy, France, China, Spain, and Australia. Finland collaborates with Sweden, France, Italy, Spain, China, the Netherlands, and the Russian Federation. Iceland’s collaborating countries are Germany, Norway, Denmark, France, the Netherlands, Finland, and Italy. Norway had top collaborations with Sweden, Denmark, Netherlands, France, Italy, Spain, and Canada. Sweden had top collaborations with Italy, France, Netherlands, China, Denmark, Spain, and Norway. It is noteworthy that the top collaborations for Iceland were all European countries. China was the only collaborating Asian country, whereas there were many collaborative publications with Denmark, Finland, and Sweden. Russia’s collaboration with Finland and Norway’s collaboration with Canada were unique. Furthermore, collaboration with Italy and Spain resulted in higher productivity and quality publications among other European countries than did the Nordic countries.\n\nFigure 4 presents the collective global collaborative publications of Nordic countries. The highest number of collaborative publications was with partners from European countries. This was followed by North America, specifically the USA. The Asia Pacific region was the third top collaborative destination, followed by the Middle Eastern region, South America, and Africa. Collaborative output from the South American and African regions was more or less equal. It is noteworthy that all five Nordic countries presented similar characteristics in terms of collaborations.\n\nCollaborative publication with industrial coauthors is also one of the important parameters indicating that translational research is happening in an academic or research institution. A country’s corporate academic collaborative publications are reliable indicators of the country’s innovation practices. More academic corporate collaborations would mean more research occurring in the direction of product development, industrial consultancy, and concept to product. An attempt is made in the present research to understand the outcomes of academic corporate collaborations in Nordic countries. Publications from 2016 to 2020 were analysed to identify the percentage of publications with international coauthors, as represented in Figure 5. As Figure 5 shows, Denmark has the highest number of coauthors from industry, approximately 10.4% of its overall publications. Iceland has 10.2%, Finland has 8.3%, Norway has 8.1%, and Sweden has 7.9% of its publications with coauthors from industry. It is important to note that the global average of academic corporate collaborative publications is 2.7%, whereas Nordic countries have an average of 8.1% of their publications with industry collaboration. Nordic countries have an almost three times higher percentage rate than the world average. This indicates that the research carried out in Nordic countries has stronger industry connections when compared to other parts of the world. This is also indicative of a large number of R&D centers in industries in Nordic countries.\n\nIn the previous sections, we have seen the collaborative outcomes of researchers in Nordic countries with researchers in other parts of the world and with industrial personnel. This section presents an analysis of the publication behavior of Nordic countries. Figure 6 shows the collaborative activities among Nordic countries. Data from smaller Nordic countries such as the Faroe Islands and Svalbard are also included. In the figure, the complete circle represents the total publications of the Nordic countries. The circle is divided into segments based on the numbers contributed by each country. Each segment is connected to the other segments using line segments. The thickness of these lines represents the number of publications. Figure 7 shows the figures for collaborative publications among Nordic countries. Denmark has the largest number of collaborative publications with Sweden, followed by Norway, Finland, and Iceland. Sweden has characteristics similar to those of Denmark, insofar as the collaboration with Denmark is highest, followed by Norway, Finland, and Iceland. Norway shows a slightly different behavior in which the highest numbers for collaborative publications are with Sweden, followed by Denmark, Finland, and Iceland. Finland has its largest number of collaborative publications with Sweden, Denmark, and Norway, with similar figures in terms of collaborations, followed by collaborations with Iceland. The highest number of collaborative publications in Iceland is with Sweden, followed by Norway, Denmark, and Finland.\n\nFigure 8 shows the results of the qualitative analysis of collaboration outcomes in terms of citations, as shown in the FWCI for collaborative publications. FWCI trends differ slightly from the numbers of published collaborative papers. Collaborative publications with Iceland have a higher average FWCI than any other collaboration among Nordic countries. This rate is followed by the figures for collaborative publications with Finland, Norway, Denmark, and Sweden. Collaborative publications have a higher FWCI than noncollaborative publications. Collaborative publications with Nordic countries also have a comparatively better FWCI than those with other parts of the world.\n\nThe quality of journals considered for publishing is one of the parameters indicative of the quality of research. Publication in top-rated journals signifies the quality of research. Figure 9 shows the output of Nordic countries in the top 25 percentage journals in their respective subject areas. Across the globe, overall, 46.1% of the papers published are in quartile one journals. However, in Nordic countries, approximately 65% of the papers published are in top quartile journals. This is a clear indication that the work produced by Nordic countries is of higher quality when compared to global trends. From the Nordic countries, approximately 69% of papers published in Denmark are in top quartile journals, followed by Sweden, with 67% of its publication in top quartile journals. Finland publishes 64.9% of its research studies in top quartile journals; 63.1% of publications from Iceland are in top quartile journals; and 62.1% of publications from Norway are in top quartile journals. This analysis clearly indicates that Nordic countries publish in high-quality journals, and because of this, the citation indexes are higher compared to global averages.\n\nJournals are classified into different categories based on the specialization in which the journal publishes articles. Each journal publishes articles from a specific field. The subject areas are classified based on the standard All Journal Science Code (AJSC). All journals are associated with one or more AJSC codes. Multidisciplinary journals are associated with more than one subject area. In the present research, an attempt is made to understand the pattern of publication from Nordic countries across different subject areas. There are approximately 250+ subcategories in the AJSC. In the current study, we consider five basic subject areas: life sciences and medicine, engineering and technology, natural sciences, social sciences and management, and arts and humanities. Figure 10 shows the distribution of publications across Nordic countries as a whole and in the five individual countries. In Nordic countries, publications in life sciences and medicine are highest at 36% of overall publications, followed by engineering and technology at 24% of overall publication numbers. Publications in natural sciences are at 21%, social sciences and management are at 15%, and publications in arts and humanities constitute approximately 4% of overall numbers.\n\nFigure 10 also shows the publication trends of individual Nordic countries in various subject areas. In all Nordic countries, publications in the area of life and medicine are highest, with 38% in Sweden, 34% in Norway, 37% in Iceland, 30% in Finland, and 43% in Denmark. This trend is followed across Nordic countries as a whole. Publications in the area of engineering and technology are the second largest, after life sciences and medicine in Sweden (24%), Norway (23%), Finland (27%), and Denmark (20%). However, the trend in Iceland is a little different, whereby publication numbers in natural sciences are second highest at 25% and those in engineering and technology are 20%. The third highest publication numbers in other Nordic countries are in natural sciences, with Sweden at 21%, Norway at 21%, Finland at 22%, and Denmark at 19%. Publications in social sciences and management are fourth highest in all Nordic countries, with 14% in Sweden, 18% in Norway, 14% in Iceland, 17% in Finland, and 13% in Denmark. The remaining number of publications are in the area of arts and humanities across all the Nordic countries. The trends are similar, with 3% in Sweden and Denmark and 4% in Norway, Finland, and Iceland.\n\nFigure 11 presents a detailed analysis with respect to citations across different subject areas in Nordic countries. The FWCI of publications from 2016 to 2021 is presented across different subject areas for the Nordic countries. As with publication numbers, the citation impact trends are similar across Nordic countries. Publications in life sciences and medicine have the highest impact score compared to all other subject areas in all the Nordic countries, with Norway at 1.84, Sweden at 1.8, Finland at 1.89, Iceland at 2.95, and Denmark at 1.92. Of all the Nordic countries, Iceland has the highest quality factor. This could be due to the large percentage of collaborative work. The second highest citation index is not like the publication numbers. The subject for which Norway has the smallest publication number (arts and humanities) has an FWCI at 1.64; Sweden is at 1.64; Finland is at 1.62; Iceland is at 1.58; and Denmark is at 1.75. The behavior of the citation index in the remainder of the subject area across Nordic countries is not uniform, as is the case with the first or second position elements. The FWCI in the area of engineering and technology is 1.44 in Norway, 1.48 in Sweden, 1.49 in Finland, 1.37 in Iceland and 1.61 in Denmark. The FWCI for natural sciences in Nordic countries is 1.48 in Norway, 1.48 in Sweden, 1.44 in Finland, 1.48 in Iceland, and 1.6 in Denmark. Finally, the FWCI in social sciences and management is 1.58 in Norway, 1.6 in Sweden, 1.58 in Finland, 1.48 in Iceland, and 1.69 in Denmark.\n\n\nConclusion\n\nThe current study was done to analyse the research outcomes of Nordic countries using Scopus, Scival and World Bank Data. The research population and research spent of different countries was compared. Among Nordic countries, Sweden, with the highest population, also has the highest researcher population. This analysis has shown that in this region, research spent and the number of researchers have shown a positive correlation with the FWCI, a citation metric that considers the differences in research behavior across different disciplines. Sweden, with the highest number of researchers, also has the highest proportion of GDP spent on research among the Nordic countries.\n\nHowever, Iceland, with a low research expenditure, has a high FWCI. On studying the collaboration within the Nordic countries, it was observed that Sweden and Denmark have the highest number of coauthored publications. Within Nordic countries, the highest FWCI is seen in coauthored publications of Iceland and Finland, closely followed by Iceland and Denmark and finally Iceland and Norway. Iceland thus shows the highest FWCI within Nordic countries despite having the smallest researcher population. All the countries in this region have a high number of coauthored publications with the United States. The only exception, again, is Iceland, which has the highest number of coauthored publications with the US, followed by Sweden. The distribution of coauthored publications across continents shows that all Nordic countries have the highest numbers of collaborations within Europe. Denmark has the highest percentage of publications in collaboration with the corporate sector, which is followed by Iceland. Denmark tops the Nordic countries with the highest number of publications in first quartile journals while Sweden ranks second. This implies that Denmark tops the list in quality publications. Life Sciences and medicine, Engineering and Technology and Natural Sciences are the top three areas in which Nordic countries are contributing. On comparing research across different subject areas, all countries have the highest number of publications in life sciences and medicine. There is also a significant contribution by Nordic countries in social sciences and management and arts and humanities. Overall, Nordic countries produce high numbers of publications across different subject areas and, in addition to global collaborations, have a large chunk of researchers who collaborate with different European nations. Having the FWCI of all the countries above the world average makes the research outcomes of this region promising. Due to the proximity of these nations and the common challenges encountered, Nordic countries, with their versatile potential in different areas of research, can work as a team to build a strong multidisciplinary research hub. This study points towards the finding that collaborative research can help in increasing the quality and thus research citations as shown in Nordic countries.\n\nThis study puts forth the research outcomes and research strengths of Nordic countries. Such information can help the researchers in different areas globally to collaborate with the researchers in these nations. The clarity about the research strength of these nations can help funders to make strategies for funding these nations. Also, this information can help other countries to follow various practices to improve the research impact.", "appendix": "Data availability\n\nOpen Science Framework: Extended data for ‘Bibliometric analysis of Nordic countries for the period 2016 to 2020’ is archived at https://doi.org/10.17605/OSF.IO/7MAZ4. 46\n\nThis project contains the following underlying data:\n\n• data_file.xlsx – Data related to bibliographic data of Nordic countries with respect to subjects.\n\n• working.xlsx – Data related to ranking and world bank parameter of Nordic countries\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nKautto M: The nordic countries. The Oxford handbook of the welfare state. 2010.\n\nEskildsen JK, Kristensen K, Westlund AH: Work motivation and job satisfaction in the Nordic countries. Empl. Relat. 2004; 26: 122–136. Publisher Full Text\n\nLane JE, Ersson S: The Nordic Countries. Political institutions in Europe. 2002; 245.\n\nKangas O, Palme J: Social policy and economic development in the Nordic countries: an introduction. Social policy and economic development in the Nordic countries. London: Palgrave Macmillan; 2005; (pp. 1–16).\n\nMartela F, Greve B, Rothstein B, et al.: The Nordic exceptionalism: what explains why the Nordic Countries are constantly among the happiest in the world.Helliwell JF, Layard R, Sachs JD, et al. editors. World Happiness Report.2020; pp. 128–145.\n\nFuru K, Wettermark B, Andersen M, et al.: The Nordic countries as a cohort for pharmacoepidemiological research. Basic Clin. Pharmacol. Toxicol. 2010; 106(2): 86–94. Publisher Full Text\n\nLin CYY, Edvinsson L: National intellectual capital: comparison of the Nordic countries. J. Intellect. Cap. 2008.\n\nMichelsen S, Stenström ML, Jørgensen CH, et al.: Vocational education in the nordic countries. Routledge; 2018.\n\nTøssebro J, Bonfils IS, Teittinen A, et al.: Normalization fifty years beyond—current trends in the Nordic countries. J. Policy Pract. Intellect. Disabil. 2012; 9(2): 134–146. Publisher Full Text\n\nLööf H, Heshmati A, Asplund R, et al.: Innovation and performance in manufacturing industries: A comparison of the Nordic countries (No. 457). SSE/EFI working paper series in economics and finance. 2001.\n\nGlänzel W: Science in Scandinavia: A bibliometric approach. Scientometrics. 2000; 48(2): 121–150. Publisher Full Text\n\nClarke A, Gatineau M, Grimaud O, et al.: A bibliometric overview of public health research in Europe. Eur. J. Pub. Health. 2007; 17(suppl_1): 43–49. PubMed Abstract \| Publisher Full Text\n\nMelin G, Persson O: Hotel cosmopolitan: A bibliometric study of collaboration at some European universities. J. Am. Soc. Inf. Sci. 1998; 49(1): 43–48. Publisher Full Text\n\nGjersvik P, Nylenna M, Jemec GB, et al.: Dermatologic research in the Nordic countries 1989–2008–a bibliometric study. Int. J. Dermatol. 2010; 49(11): 1276–1281. PubMed Abstract \| Publisher Full Text\n\nSchneider JW: Bibliometric Research Performance Indicators for the Nordic Countries: A publication from the NORIA-net “The use of bibliometrics in research policy and evaluation activities”.2010.\n\nSandnes FE: A bibliometric study of human–computer interaction research activity in the Nordic-Baltic Eight countries. Scientometrics. 2021; 126(6): 4733–4767. Publisher Full Text\n\nGlänzel W, Schlemmer B, Schubert A, et al.: Proceedings literature as additional data source for bibliometric analysis. Scientometrics. 2006; 68(3): 457–473. Publisher Full Text\n\nKoskinen J, Isohanni M, Paajala H, et al.: How to use bibliometric methods in evaluation of scientific research? An example from Finnish schizophrenia research. Nord. J. Psychiatry. 2008; 62(2): 136–143. PubMed Abstract \| Publisher Full Text\n\nTarkowski SM: Environmental health research in Europe–bibliometric analysis. Eur. J. Pub. Health. 2007; 17(suppl_1): 14–18. Publisher Full Text\n\nRabow H: Bibliometric research output indicators and university funding in the Nordic countries. ScieCom Info. 2012; 8(1).\n\nMerigó JM, Gil-Lafuente AM, Yager RR: An overview of fuzzy research with bibliometric indicators. Appl. Soft Comput. 2015; 27: 420–433. Publisher Full Text\n\nHanvold TN, Kines P, Nykänen M, et al.: Occupational safety and health among young workers in the Nordic countries: a systematic literature review. Saf. Health Work. 2019; 10(1): 3–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWidfeldt A: The radical right in the Nordic Countries. The Oxford handbook of the radical right. 2018.\n\nKnudsen JP: Dealing with rural–urban economic welfare challenges in the Nordic countries–a theory-based overview. Nordisk välfärdsforskning\|Nordic Welfare Research. 2020; 5(1): 58–69. Publisher Full Text\n\nThomsen JP, Bertilsson E, Dalberg T, et al.: Higher education participation in the Nordic countries 1985–2010—a comparative perspective. Eur. Sociol. Rev. 2017; 33(1): 98–111.\n\nAirey J, Lauridsen KM, Räsänen A, et al.: The expansion of English-medium instruction in the Nordic countries: Can top-down university language policies encourage bottom-up disciplinary literacy goals? High. Educ. 2017; 73(4): 561–576. Publisher Full Text\n\nSkogerbø E, Ihlen Ø, Nörgaard Kristensen N, et al.: Power, communication, and politics in the Nordic countries. Nordicom.2021.\n\nChristensen S, Zhang W: Shadow Education in the Nordic Countries: An Emerging Phenomenon in Comparative Perspective. ECNU Rev. Educ. 2021; 4(3): 431–441. Publisher Full Text\n\nTikkanen T: Problem-solving skills, skills needs and participation in lifelong learning in technology-intensive work in the Nordic countries. Journal of Contemporary Educational Studies/Sodobna Pedagogika. 2017; 68(4).\n\nBaccini A, De Nicolao G, Petrovich E: Citation gaming induced by bibliometric evaluation: A country-level comparative analysis. PLoS One. 2019; 14(9): e0221212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLópez-Illescas C, de Moya Anegón F , Moed HF: Comparing bibliometric country-by-country rankings derived from the Web of Science and Scopus: The effect of poorly cited journals in oncology. J. Inf. Sci. 2009; 35(2): 244–256. Publisher Full Text\n\nZacca-González G, Chinchilla-Rodríguez Z, Vargas-Quesada B, et al.: Bibliometric analysis of regional Latin America’s scientific output in public health through SCImago journal & country rank. BMC Public Health. 2014; 14(1): 1–11. Publisher Full Text\n\nKhiste GP, Paithankar RR: Analysis of Bibliometric term in Scopus. International Journal of Library Science and Information Management (IJLSIM). 2017; 3(3): 81–88.\n\nTibaná-Herrera G, Fernández-Bajón MT, de Moya Anegón F : Output, collaboration and impact of e-learning research: Bibliometric analysis and visualizations at the country and institutional level (Scopus 2003-2016). Profesional de la Información. 2018; 27: 1082. Publisher Full Text\n\nShehatta I, Al-Rubaish AM: Impact of country self-citations on bibliometric indicators and ranking of most productive countries. Scientometrics. 2019; 120(2): 775–791. Publisher Full Text\n\nHassan-Montero Y, Guerrero-Bote VP, De-Moya-Anegón F: Graphical interface of the Scimago Journal and Country Rank: an interactive approach to accessing bibliometric information. El profesional de la información. 2014; 23(3): 272–278. Publisher Full Text\n\nArchambault É, Campbell D, Gingras Y, et al.: Comparing bibliometric statistics obtained from the Web of Science and Scopus. J. Am. Soc. Inf. Sci. Technol. 2009; 60(7): 1320–1326. Publisher Full Text\n\nLin G, Hu Z, Hou H: Research preferences of the G20 countries: a bibliometrics and visualization analysis. Curr. Sci. (00113891). 2018; 115(8): 1477. Publisher Full Text\n\nCorrall S, Kennan MA, Afzal W: Bibliometrics and research data management services: Emerging trends in library support for research. Libr. Trends. 2013; 61(3): 636–674. Publisher Full Text\n\nMiremadi I, Saboohi Y, Arasti M: The influence of public R&D and knowledge spillovers on the development of renewable energy sources: The case of the Nordic countries. Technol. Forecast. Soc. Chang. 2019; 146: 450–463. Publisher Full Text\n\nDe la Porte C, Jensen MD, Kvist J: Going Nordic—Can the Nordic model tackle grand challenges and be a beacon to follow? Regul. Gov. 2023; 17(3): 595–607. Publisher Full Text\n\nKarseth B, Sivesind K, Steiner-Khamsi G: Evidence and expertise in Nordic education policy: A comparative network analysis. Springer Nature; 2022; p. 429. Publisher Full Text\n\nVerger A: Evidence-Based Policy Making and Educational Reform in Nordic Europe: Key Contributions of the POLNET Study. Evidence and Expertise in Nordic Education Policy: A Comparative Network Analysis. Cham: Springer International Publishing; 2022; pp. 395–408. Publisher Full Text\n\nAndersson C, Sund K: Technical Efficiency and Productivity of Higher Education Institutions in the Nordic Countries. Int. J. Public Adm. 2022; 45(2): 107–120. Publisher Full Text\n\nSivesind K, Karseth B: Introduction: A Comparative Network Analysis of Knowledge Use in Nordic Education Policies. Evidence and Expertise in Nordic Education Policy: A Comparative Network Analysis. Cham: Springer International Publishing; 2022; pp. 1–31. Publisher Full Text\n\nSanthosh KV, Janodia M, Magazine BC, et al.: Nordic countries [Dataset.] 2023, April 29. Publisher Full Text" }	[ { "id": "276617", "date": "08 Jun 2024", "name": "Mats Benner", "expertise": [ "Reviewer Expertise Research policy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper affords a descriptive overview of research conditions in the Nordic countries, citing the relevant sources and providing information in an attainable and accessible form. What it does not do is to afford an analysis of the Nordic countries beyond that, i.e. how the composition of the research systems have evolved time time, why collaborative patterns take the form they take, and what overall conclusions can be drawn. There is an abundance of literature on the evolution of research system, how funding arrangements are aligned with over policy priorities, and forms of implementation at the level of universities, companies and institutes, and similar. Some of the literature deal with overall steering principles for research (i.e. principal-agent, systems of innovation, governance models, etc), others with the historical and comparative development of institutional arrangements and the relationship between inputs (funding primarily) and outputs (publications, citations, patents). The text does not engage with these literatures. What it does is to present an overall descriptive account of the state of research in the Nordics based on openly available statistics. This means that its contributions are exclusively at the level of description, not theory or 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? 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": "297294", "date": "10 Jul 2024", "name": "Palanisamy Sivanandy", "expertise": [ "Reviewer Expertise Medical Science", "Health and Life 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\n1. What is the rationale for comparing the country's population and the number of researchers? any significant findings using these values? must be explained in the abstract and discussion section. 2. It is good to explain the data extraction using a structured flowchart with the number of articles from every source like PubMed, Science Direct, Cochrane, etc. 3. The statistical tests applied in this research are not included fully. In the method section, only MS Excel was mentioned, however, it is better to add a correlation analysis of publications with different countries and impact factor/citation indexes. 4. Figure 3, compares the research spending by percentage of GDP among the Nordic countries.\" How GDP was compared with the research spending? need to be explained in the method/results section. 5. Why data from the Faroe Islands and Greenland were excluded? Explain the reasons. 6. Throughout the article it was mentioned as more publications on life sciences. Life science is a broad terminology, it is better to segregate the articles within the subsection/class of life sciences.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "12113", "date": "31 Jul 2024", "name": "Santhosh Venkata", "role": "Author Response", "response": "What is the rationale for comparing the country's population and the number of researchers? any significant findings using these values? must be explained in the abstract and discussion section. Response: There is an uneven distribution of researchers in the world. By comparing, the number of researchers with population gives an idea about the percent growth of researcher’s pool when the country’s population is increasing. We attempted to understand researcher per 100000 population in these countries and its correlation with the % of GDP spend on research. Hence, we compared population with researchers. The rationale is explained in paragraph above fig 2. It is good to explain the data extraction using a structured flowchart with the number of articles from every source like PubMed, Science Direct, Cochrane, etc. Response: Since this is a bibliometric study, the data was extracted from databases like Scopus and SciVal, which are products from Elsevier. For this study, we did not use PubMed, ScienceDirect or Cochrane as bibliometric data cannot be accessed from these databases. The statistical tests applied in this research are not included fully. In the method section, only MS Excel was mentioned, however, it is better to add a correlation analysis of publications with different countries and impact factor/citation indexes. Response: With respect to the objectives of the study, there are no statistical tests applied. The bibliometric data from Scopus and SciVal was extracted in the form of excel sheet for analysis. There is no correlation studied in this manuscript. The impact factor as a metric, is used for journals and not at the country level. In the bibliometric studies the accepted citation index is FWCI, which we have mentioned in the manuscript. However, added Correlation of Countries with bibliographic metrics (Scholarly Output, Citation, Citation per Publication, FWCI) and Correlation of co-authored publication among countries Figure 3, compares the research spending by percentage of GDP among the Nordic countries.\" How was GDP compared with the research spending? need to be explained in the method/results section. Response: As per the World Bank Data, the research spent is considered as percentage of GDP. So we have shown in figure 3 the relation of research spent with respect to FWCI. Research spent percentage gives a fair idea of ruling governments commitment to research growth Why data from the Faroe Islands and Greenland were excluded? Explain the reasons. Response: Most literature on Nordic countries emphasize on prominent five countries studied. Further, Faroe Islands is an autonomous region governed by Denmark, which is already studied. Moreover, these are small islands with maximum of 60,000 population with one university in each of the islands. Also, the population in this area is predominantly migratory and thus this area will not give a correct dynamic of research productivity in alignment with other countries. Throughout the article it was mentioned as more publications on life sciences. Life science is a broad terminology, it is better to segregate the articles within the subsection/class of life sciences. Response: The term “Life Science” is a category identified by the Scopus and SciVal. Since, this is a broad term used to describe in Scopus, along with other broad domains based on ranking agencies (five broad categories for the QS and 11 categories for THE), we have refrained from further subclassification of the term life science." } ] } ]	1	https://f1000research.com/articles/13-71
https://f1000research.com/articles/9-718/v1	17 Jul 20	{ "type": "Research Article", "title": "Mental health, sleep quality and quality of life in individuals with and without multiple health conditions during home quarantine in India due to the COVID-19 pandemic: a cross-sectional study", "authors": [ "Ramesh Chandra Patra", "Biswajit Kanungo", "Parul Bawa", "Biswajit Kanungo", "Parul Bawa" ], "abstract": "Background: Since the World Health Organization declared the COVID-19 outbreak a global pandemic, global spread has created several challenges for the general public and health care workers across the world. The primary aim of this study was to assess the psychological stress, sleep quality, and health-related quality of life (QoL) of individuals with multiple health issues during home quarantine caused by the COVID-19 pandemic. Methods: 50 individuals were recruited between 28th March and 30th April 2020, who have a history of chronic health issues, and 50 individuals with no health issues for this cross-sectional study. Three questionnaires were used to evaluate the mental health [depression anxiety stress scale (DASS-21)], sleep quality [Pittsburgh sleep quality index (PSQI)], and QoL [short form of health-related questionnaire (SF-36)] of participants. Statistical analysis was carried out with Student’s t-test, using SPSS software v16. Results: Baseline demographic characteristics were homogenous for both groups of participants. Intergroup analysis revealed statistically significant differences in mental health (p<0.001), sleep quality (p<0.001), and QoL (p<0.001) between the two groups. Conclusion: Our findings indicate that individuals with chronic health issues exhibit higher mental health problems, lower quality of sleep and have a lower health-related QoL. More research needs to be done for this group of individuals and the Government should plan to care of these individuals.", "keywords": [ "COVID-19", "Mental Health", "Sleep Quality", "Quality of Life" ], "content": "Introduction\n\nAccording to the World Health Organization (WHO), the COVID-19 outbreak is a global pandemic that started at the end of November in China and then gradually spread all over the world1,2. As neither a treatment nor vaccine have been discovered for this disease, it raises concerns among the public about the spread of infection from confirmed COVID-19 positive cases. The WHO suggests that social isolation helps to limit the growing number of cases of COVID-19, and this has also led to significant fear and anxiety related to the spread of infection in the general public. Excessive fear and apprehension about the spread of infection can lead to acute stress, anxiety, and low quality of sleep3,4. Many organizations are already working to increase awareness about the societal impact of the on-going pandemic5. For instance, it has been reported that during this pandemic crisis, various factors that impact the health of inviduals, such as prolonged periods of social isolation, fear of unemployment and economic loss, have the potential to increase due to lockdown5–7.\n\nThe relationship between physical illness and mental health has received increased attention in recent years, and poor mental health is of concern as it may exert a negative effect on an individual's quality of life (QoL)8,9. Evidence suggests that there is a direct relationship between mental health and sleep quality, a crucial public health issue10,11. In this study, we aimed to assess thepsychological stress, sleep quality, and health-related QoL of individuals with and without multiple health issues during home quarantine in India due to the COVID-19 pandemic.\n\n\nMethods\n\nDuring the conduct of the study, all human ethical principles as per the World Medical Association’s Declaration of Helsinki (2013) and the guidelines of Good Clinical Practice (Indian Council of Medical Research) were observed.\n\nWe obtained approval for the study from the Institutional Research Ethics Committee of the Lovely Professional University (LPU), Phagwara, India (LPU/IEC/2020/26/03). All the participants were informed about the procedure of data collection, and a written consent form was obtained from all the participants prior to the study start.\n\nThis study was an observational cross-sectional study. The study took place at the Department of Physiotherapy, LPU. The study was conducted from 28th March to 30th April 2020. Full lock down in India was initiated from 28th March to 31st May 2020. From 31st May, the lockdown was extended until 30th June for certain containment zones.\n\nA total of 100 participants were recruited for the study. A total of 50 individuals suffering from chronic health issues (Group A; as identified from their clinical records) and 50 individuals with no chronic health issues (Group B) were recruited.\n\nParticipants were recruited from individuals undergoing physio treatment at the Department of Physiotherapy, LPU (even though lockdown was ongoing, essential services, such as physio appointments, were ongoing).\n\nUsing the convenience sampling method, individuals were contacted via phone to ask if they would like to take part in the study. If the individual consented, in their next physio treatment appointment, they were asked to sign the informed consent form and fill in the three questionnaires (as below).\n\nGroup A inclusion criteria: clinically pre-diagnosed with hypertension, diabetics, and chronic musculoskeletal conditions.\n\nExclusion criteria Groups A and B: history of any malignancy, recent fracture or trauma, osteoporosis, inflammatory arthritis, and/or cauda equine syndrome.\n\nThere were no inclusion criteria for Group B.\n\nMental health. Depression, anxiety, and stress scale (DASS-21)12 was used to assess depression, anxiety and stress. The participants were asked to utilize a four-point severity/frequency scale to show the level of depression, anxiety and stress they were experiencing in the past week. The scale consists of 21 questions with three subscales of depression, stress, and anxiety and each subscale comprises seven questions each. Each subscale comprises of seven statements regarding how the test subject was feeling over the last week and four responses ranging from 0- did not apply to me at all, 1- applied to me some of the time, 2- applied to me for a considerable amount of time to 3- applied to me very much/most of the time. The total score for each subscale gives the severity of that very symptom which has a range from 0 to 21 for each subscale.\n\nSleep quality. Sleep quality was evaluated through the Pittsburgh Sleep Quality Index (PSQI)13. This index asked participants to answer questions about their sleep habits in the past month. Participants that scored more than 5 were defined as having a low sleep quality.\n\nQuality of life. Health-related QoL was assessed using the MOS 36-item short-form health survey (SF-36)14. The 36 items reflect eight health-related aspects that participants are asked to score, where 100 is defined as perfect health less, and any score less than 100 is defined as poor health.\n\nBaseline characteristics of categorical variables were evaluated using Chi-square test. Quantitative variables were evaluated using Student's t-test, and quantitative variables without normal distribution were measured using the Mann-Whitney U test. Intergroup outcome measures were evaluated through an unpaired t-test. All analyses were carried out on SPSS software v16.\n\n\nResults\n\nA total of 110 participants were selected for primary assessment; 10 individuals were excluded as they did not fulfil the inclusion criteria. In total, 50 participants with chronic health issues and 50 without health issues were evaluated for the study.\n\nDemographic characteristics are shown in Table 1. There was no statistical difference between groups for demographic characteristics.\n\nGroup A, individuals with chronic health issues; Group B, individuals without chronic health issues. Socioeconomic status was calculated as follows: Poor, below Rs 15000/month; average, Rs 15000–100000/month; high, above Rs 100000/month.\n\nPF: physical functioning, RL-PH: role of limitation-physical health, RL-EH: role of limitation-emotional health, EN: energy, EWB: emotional wellbeing, BP: Body pain, GH: general health, DASS=Depression Anxiety Stress Scale, PSQI= Pittsburgh Sleep Quality Index, *Social life (SL) domain was not considered for this study.\n\nGroup A, individuals with chronic health issues; Group B, individuals without chronic health issues.\n\nTable 2 presents the scores for Groups A and B for the three outcome measures (mental health, sleep quality and health-related QoL). For all DASS-21 items (mental health), Group A scored higher than Group B, showing higher levels of depression, anxiety and stress in Group A individuals. Similarly, for PSQI, Group A scored higher than Group B, showing poorer sleep quality for Group A individuals. For all SF-26 items, Group A scored lower than Group B, revealing lower health-related QoL in Group A individuals. Unpaired t-tests showed statistically significant differences between the groups for all variables (p = 0.001).\n\n\nDiscussion\n\nSince the WHO declared the COVID-19 outbreak a global pandemic, many individuals, even those who have not been infected by the virus, are required to follow government rules where it was mandatory to stay at home. In this cross-sectional study, we sought to identify the correlation between chronic health issues and depression, anxiety, stress, quality of sleep and QoL in a population-based study in India during lockdown due to COVID-19. Our findings showed that poor mental health, low sleep quality, and low health-related QoL were higher in individuals with chronic health issues compared with individuals without chronic health issues at the time of home quarantine in India.\n\nCurrent evidence reveal that COVID-19 causes fear among the Indian population as they are in home quarantined due to lockdown, which can an impact on well-being, increasing the levels of depression, anxiety, stress, reducing sleep quality, and decreasing QoL15,16.\n\nConsidering that lockdown is likely to last for weeks, there is an urgent need to monitor the psycho-physiological well-being of the population and to collect research data to develop evidence-based strategies to reduce negative psychological effects of these unprecedented changes in individuals’ everyday lives17,18, especially in those with chronic health conditions.\n\nThis study has some potential limitations: participants were recruited in only one area of India, we had a small sample size, and only subjective outcome was considered for the study\n\n\nConclusion\n\nThe WHO has recommended that individuals with physical and mental disabilities need to take extra care during isolation/quarantine for COVID-1919. This is supported by the results of this study, which revealed that there is an increased level of depression, anxiety, stress, and reduced sleep quality and QoL shown in individuals suffering from chronic illness compared with individuals without chronic illness during the quarantine period in India.\n\n\nData availability\n\nFigshare: Mental Health, Sleep Quality and Quality of Life in Subjects with Multiple Health Conditions during Home Quarantine for COVID-19 Pandemic Attack: A Comparison with Healthy Subjects, https://doi.org/10.6084/m9.figshare.1261283320.\n\nThis project contains the following underlying data:\n\n- Group A data\n\n- Group B data\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe author would like to express heartfelt thanks to all the participants who were participated in this study.\n\n\nReferences\n\nCucinotta D, Vanelli M: WHO declares COVID-19 a pandemic. Acta Biomed. 2020; 91(1): 157–60. PubMed Abstract \| Publisher Full Text\n\nCascella M, Rajnik M, Cuomo A, et al.: Features, evaluation and treatment coronavirus (COVID-19). InStatpearls. [internet] StatPearls Publishing. 2020. PubMed Abstract\n\nChen H, Guo J, Wang C, et al.: Clinical characteristics and intrauterine vertical transmission potential of COVID-19 infection in nine pregnant women: a retrospective review of medical records. Lancet. 2020; 395(10226): 809–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Pan R, Wan X, et al.: Immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (COVID-19) epidemic among the general population in China. Int J Environ Res Public Health. 2020; 17(5): 1729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahoo S, Bharadwaj S, Parveen S, et al.: Self-harm and COVID-19 Pandemic: An emerging concern–A report of 2 cases from India. Asian J Psychiatr. 2020; 51: 102104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoyal K, Chauhan P, Chhikara K, et al.: Fear of COVID 2019: First suicidal case in India! Asian J Psychiatr. 2020; 49: 101989. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLAW K: COLLEGE Approved by Bar Council of India and affiliated to Karnataka State Law University, Hubli. KLE Society.\n\nBiddle SJ, Ciaccioni S, Thomas G, et al.: Physical activity and mental health in children and adolescents: An updated review of reviews and an analysis of causality. Psychol Sport Exerc. 2019; 42: 146–55. Publisher Full Text\n\nFox KR: The influence of physical activity on mental well-being. Public Health Nutr. 1999; 2(3a): 411–8. PubMed Abstract \| Publisher Full Text\n\nBaglioni C, Battagliese G, Feige B, et al.: Insomnia as a predictor of depression: a meta-analytic evaluation of longitudinal epidemiological studies. J Affect Disord. 2011; 135(1–3): 10–9. PubMed Abstract \| Publisher Full Text\n\nSpira AP, Covinsky K, Rebok GW, et al.: Poor sleep quality and functional decline in older women. J Am Geriatr Soc. 2012; 60(6): 1092–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe MT, Tran TD, Holton S, et al.: Reliability, convergent validity and factor structure of the DASS-21 in a sample of Vietnamese adolescents. PLoS One. 2017; 12(7): e0180557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHita-Contreras F, Martínez-López E, Latorre-Román PA, et al.: Reliability and validity of the Spanish version of the Pittsburgh Sleep Quality Index (PSQI) in patients with fibromyalgia. Rheumatol Int. 2014; 34(7): 929–36. PubMed Abstract \| Publisher Full Text\n\nSchlenk EA, Erlen JA, Dunbar-Jacob J, et al.: Health-related quality of life in chronic disorders: a comparison across studies using the MOS SF-36. Qual Life Res. 1997; 7(1): 57–65. PubMed Abstract \| Publisher Full Text\n\nSood S: Psychological effects of the Coronavirus disease-2019 pandemic. Research & Humanities in Medical Education. 2020; 7: 23–6. Reference Source\n\nKazmi SS, Hasan K, Talib S, et al.: COVID-19 and Lockdwon: A Study on the Impact on Mental Health. Available at SSRN 3577515. 2020. Publisher Full Text\n\nLazzerini M, Putoto G: COVID-19 in Italy: momentous decisions and many uncertainties. Lancet Glob Health. 2020; 8(5): e641–e642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Mental health and psychosocial considerations during the COVID-19 outbreak, 18 March 2020. World Health Organization; 2020. Reference Source\n\nPatra RC, kanungo B, Bawa P: Mental Health, Sleep Quality and Quality of Life in Subjects with Multiple Health Conditions during Home Quarantine for COVID-19 Pandemic Attack: A Comparison with Healthy Subjects. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12612833.v1" }	[ { "id": "73792", "date": "13 Nov 2020", "name": "Shyamal Koley", "expertise": [ "Reviewer Expertise Kinanthropometry", "Biomechanics", "Public Health & Lectinology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Abstract, Introduction, Methods, Results, & Discussion Portions of the article are written nicely. The only correction needed is in Acknowledgments where \" The author would......\" should be written as \" The authors would......\". References portion are written as per the style of the journal and adequate and expressive. The article is timely written and carries sufficient applicability.\nDetailed Report:\nCOVID-19 is a serious global pandemic which affects physical, mental, social and societal aspects of individuals of all strata of the society. The present study attempted justly in this regard. It is a very recent complication, thus not much literature would be available. Considering the above fact, the literature cited in the article was justified. It was quite difficult to collect the samples in the time of Lockdown prevailed then nationwide. Still the work would be considered as technically sound. In such type of study, the controls are not necessarily required, pre- and post-conditions may work. The methods and analyses were sound with proper replicability. Though the sample size was small (n=100), statistical analyses (using Chi-square test, Student’s t-test and Man-Whitney U test) were appropriate. The conclusion drawn in the study was adequately supported by the results. Small sample size was one of the limitations of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6117", "date": "13 Nov 2020", "name": "Ramesh Patra", "role": "Author Response", "response": "Thank You so much for your Valuable response and comments." } ] }, { "id": "82045", "date": "08 Apr 2021", "name": "Kanwar Hamza Shuja", "expertise": [ "Reviewer Expertise Public Health", "Criminal Psychology", "Clinical 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\nFirstly thank you for giving me the chance for reviewing the article. It's a well written article though there are places where some sentences needs to be rewritten and checked for grammar. However, my biggest concern with this article is regarding its sample size. My first question is how did authors come up with a sample size of 50? Did they selected it themselves or what was the reason behind it or did they used sampling size calculator. Because a sample of 50 is too low for concluding this study.\nSecondly, again with the sample but this time inclusion criteria for the group B was not set by the authors. Having no inclusion criteria could lead to potentially adding participants who may also be medically ill, or were these participants were inquired prior during recruitment if they are medically sound. And if they were asked that would have become their inclusion criteria. But unfortunately, I was unable to find any such thing in demographic information.\nThird the instruments used are for measuring psychological variables in general. What I mean by this is that nowhere was I able to find anything where the participants were asked their thoughts on Covid-19 or its effect on them. Now depression, stress or anxiety could be potentially due to multiple factors same with the reasons for disturbed quality of life or sleep. The authors should have added some questions in demographic which could help the participants and the readers to know that the findings here are actually due to Covid-19 and not due to any external factor.\nFourth it would would be nice if the authors can add limitations and recommendations heading before conclusion. Thank you and good luck.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "8383", "date": "07 Sep 2022", "name": "Ramesh Patra", "role": "Author Response", "response": "First, we would like to thank the honourable reviewers and editor for their constructive criticisms and suggestive comments, which were of great help in improving the quality and clarity of the current manuscript. We have read each reviewer’s comments carefully and, accordingly, made utmost efforts to address each comment point-by-point. We much appreciate the reviewer’s time, effort, and detailed explanations. We believe that the revised version of our manuscript is now more focused, scientific, understandable, and better arranged. All changes are highlighted with red color. Reviewer Comment: Query 1: Firstly thank you for giving me the chance for reviewing the article. It's a well written article though there are places where some sentences needs to be rewritten and checked for grammar. Response: Thank you for your valuable comments. We have modified the text with proper grammar usage as per your suggestions. Query 2: However, my biggest concern with this article is regarding its sample size. My first question is how did authors come up with a sample size of 50? Did they selected it themselves or what was the reason behind it or did they used sampling size calculator. Because a sample of 50 is too low for concluding this study. Response: The total sample taken was 100. We have calculated the sample size using G power software 3.1 which was found to be around 51 (95% confidence interval, power of the study was 80% where the effect size was considered as 0.5). The sample size calculation has been added in the revised manuscript under the method section. Query 3: Secondly, again with the sample but this time inclusion criteria for the group B was not set by the authors. Having no inclusion criteria could lead to potentially adding participants who may also be medically ill, or were these participants inquired prior during recruitment if they are medically sound. And if they were asked that would have become their inclusion criteria. But unfortunately, I was unable to find any such thing in demographic information. Response: As suggested, we have included the inclusion criteria for Group B. Query 4: Third, the instruments used, are for measuring psychological variables in general. What I mean by this is that nowhere was I able to find anything where the participants were asked their thoughts on Covid-19 or its effect on them. Now depression, stress or anxiety could be potentially due to multiple factors same with the reasons for disturbed quality of life or sleep. The authors should have added some questions in demographic which could help the participants and the readers to know that the findings here are actually due to Covid-19 and not due to any external factor. Response: We have used the following tools to measure all the three measure outcomes. DASS-21 tool was used to measure the mental health. PSQI was used to measure sleep quality. MOS-36 was used to measure the health-related QoL. We have mentioned the tools in the methodology section and also given references. Query 5: Fourth it would be nice if the authors can add limitations and recommendations heading before conclusion. Response: We have included the limitations and recommendations as per your suggestions." } ] } ]	1	https://f1000research.com/articles/9-718
https://f1000research.com/articles/13-49/v1	08 Jan 24	{ "type": "Study Protocol", "title": "A protocol for a comparative evaluation of the fracture resistance of endodontically treated teeth reinforced with Cention N, resin-modified glass ionomer cement (RMGIC) and short fiber reinforced flowable composite as an intraorifice barrier", "authors": [ "Namrata Jidewar", "Manoj Chandak", "Manoj Chandak" ], "abstract": "Background: Endodontic treatment is the most common method for resolving pulpal and periapical pathology. However, various studies have reported that almost 11%–13% of all teeth that undergo extraction after endodontic treatment show the presence of cracks, craze lines, and vertical root fractures. Teeth with inadequate post endodontic restoration are more prone to fracture and coronal leakage, resulting in the diffusion of oral fluids, bacteria, bacterial products, and possibly root canal treatment failure. Furthermore, studies have advocated the use of endodontically treated teeth with restorative materials that have a similar or higher elastic modulus than the tooth for providing stiffness against forces that cause root fracture. Intraorifice barriers made of restorative materials that can bond to radicular dentin could thus be used to reinforce the radicular dentin while also preventing coronal microleakage. Although the sealing ability of intraorifice barriers has been widely compared in the literature, there have been few studies on the strengthening effect of the materials used in the study as intraorifice barriers when placed into the root canal. As a result, the current in vitro study aims to assess the effect of various materials as intraorifice barriers (Cention N, Resin modified glass ionomer cement, and short fiber reinforced flowable composite) on the force required fracture teeth after root canal treatment. Methods: This in vitro study will be done on extracted human mandibular premolars with single root canal where after doing root canal treatment 2-3 mm obturating material would be replaced by intra orifice barriers (Cention N, resin modified glass ionomer cement [RMGIC], and short fiber reinforced flowable composite). The force required to fracture teeth will be calculated using universal testing machine.", "keywords": [ "dentistry", "endodontics", "Intraorifice barrier", "post endodontic restoration", "“Cention N”", "“Resin modified glass ionomer cement”", "“short fiber reinforced flowable composite”" ], "content": "Introduction\n\nEndodontic treatment is the primary approach for the resolution of pulpal and periapical pathology. However, various studies have reported that almost 11%–13% of all teeth that undergo extraction after endodontic treatment show the presence of cracks, craze lines, and vertical root fractures.1 It is well-known that teeth with inadequate post endodontic restoration are more prone to fracture and coronal leakage, causing diffusion of oral fluids, bacteria, bacterial products, and possibly root canal treatment failure.2 Irrigating solutions and intra canal medicaments used during chemico-mechanical preparation of root canal can alter collagen structure, which contributes to alteration of mechanical properties of dentin, resulting in fatigue crack propagation and hence increasing its susceptibility to vertical root fracture.2 The concept of intraorifice barrier placement was first given by Roghanizad and Jones for prevention of microleakage.3 Roghanizad and Jones, in order to reduce leakage, first proposed replacement of 3-mm of gutta-percha with restorative material at the root canal orifice.1 Moreover, studies have advocated the use of restorative materials for endodontically treated teeth which have a similar or higher elastic modulus than the tooth can be proposed for providing stiffness against forces that generate root fracture.1 Thus, intraorifice barriers with restorative materials that could bond to radicular dentin could be used for additionally reinforcing the radicular dentin as well as preventing coronal microleakage. Although the intra-orifice barriers was compared in terms of sealing ability in the literature widely, however there are limited studies in respect to strengthening effect of the materials which are being included in the study as intra orifice barriers when placed into the root canal.4\n\nAs a result, the current study aims to assess the effect of various materials as intraorifice barriers (Cention N, Resin modified glass ionomer cement, and short fibre reinforced flowable composite) on the force required fracture teeth after root canal treatment.\n\n\nObjectives\n\n\n\n1. To evaluate “fracture resistance” of endodontically treated teeth reinforced with Cention N as an intra orifice barrier.\n\n2. To evaluate “fracture resistance” of endodontically treated teeth reinforced with RMGIC (resin modified glass ionomer cement) as an intra orifice barrier.\n\n3. To evaluate “fracture resistance” of endodontically treated teeth reinforced with short fiber reinforced flowable composite as an intra orifice barrier.\n\n4. To compare “fracture resistance” of endodontically treated teeth reinforced with Cention N, RMGIC, short fiber reinforced flowable composite as an intra orifice barrier.\n\nStudy setting\n\nThis in vitro study will be performed in the department of conservative dentistry and endodontics at Sharad Pawar Dental College in collaboration with central research laboratory (centre of translational sciences), Sawangi wardha.\n\nSample selection\n\nThis is an in vitro study and the sample required are human mandibular premolar extracted teeth. The patients whose treatment plan would include extraction of the teeth will be prior informed about the use of their extracted teeth for the study purpose and consent will be taken.\n\nInclusion criteria\n\n• Teeth freshly extracted for orthodontic reasons with single canal and <10° curvature\n\n• Noncarious human mandibular premolar teeth will be selected\n\n• A vernier caliper will be used to measure the mesiodistal and buccolingual diameters of coronal aspect. A deviation up to ±10% from these mean values (mean = sum of all the values of mesio distal diameter of teeth/total number of extracted teeth to be used in the study) obtained will be considered as the sample\n\nExclusion criteria\n\n• Teeth with cracks or crack lines from stereomicroscope (stereo zoom microscope ASI-ZOOM-I) evaluation (10×) will be discarded\n\n• Teeth with more than one canal, existing caries, open apices, and curvature of roots more than 10° and with mesiodistal and buccolingual diameter of coronal plane exhibiting more than 10% difference from average will be excluded\n\n• Radiographic assessment will be taken of the extracted teeth and it will be checked to ensure that the tooth has one canal. Teeth having internal and/or external resorption will be excluded\n\n\n\n1. Resin modified glass ionomer cement (3M EspeVitremer) distributor – ayushi dentistry catalogue number – AD00366\n\n2. Cention N (an alkasite group restorative material) (ivoclar viva dent) distributor – dentganga, catalogue number – DGIN21091-7\n\n3. Short fiber reinforced flowable composite (Gc EverX Flow) distributor – get me dental, catalogue number – GCEVERXFO01\n\n4. Diadent dia-proseal root canal sealer distributor – dentganga, catalogue number – DGIN220122-5947\n\nGroup 1 - Cention N\n\nGroup 2 - Resin modified glass ionomer cement\n\nGroup 3 - short fiber reinforced flowable composite\n\nFor sample preparation, 33 intact mandibular premolar human teeth of similar dimensions will be selected for this study.\n\nSpecimens will be standardized to the length of 14 mm using a measuring scale. and decoronated by using diamond disc and water coolant. Patency and working length to be determined by #10K file. The canals will be biomechanically prepared with “rotary ProTaper Universal system (Dentsply)” till F3 using the crown-down technique (it is a technique of root canal preparation).5\n\nDuring instrumentation, canal irrigation will be done with 2 ml of 5% NaOCl and distilled water after each file change and lastly with a rinse of 5 ml of 17% EDTA. Obturation will be performed using the single cone technique with corresponding gutta-percha points and diadent dia-proseal sealer.\n\nThe teeth will be assigned to four groups (n = 11) by random sampling for the placement of intraorifice barriers. Except for the control group, all specimens will be prepared by removing coronal 3 mm of gutta-percha with a spoon excavator heated on a Bunsen burner.\n\nGroup 1: 4-Cention N (ivoclar) powder and liquid will be manipulated according to manufacturer’s instructions and placed in the cavity. It will be light-cured (woodpecker Light cure LED Mini-S) for 30 s.\n\nGroup 2: RMGIC (Vitremer, 3M ESPE, USA), Shade A3. The primer will be applied with an applicator tip for 30 s to dentin and air dried. The primer will be then light cured for 20 s. After manipulating according to the manufacturer’s instructions, the material will be placed into the cavity, condensed and light-cured for 40 s.\n\nGroup 3: Short fiber reinforced flowable composite (Gc EverX Flow). The primer will be applied with a brush for 30 s to dentin and air dried. The primer will be then light cured for 20 s. the composite will be then placed into the cavity and light cured for 20 s.\n\nAfter placing the intraorifice barrier materials, all specimens will be stored at 37°C and at 100% humidity for 1 week in an incubator. The apical root end of each tooth will be mounted vertically along the long axis in self-curing acrylic resin such that 3 mm of each root will be exposed.\n\nIn order to acquire periodontal ligament simulation as in a tooth socket, light body elastomeric impression material will be used between the acrylic mold surface and tooth.\n\nThe specimens will be mounted on a Computerized Universal Testing Machine mechatronic UTE-20 (mechatronic in CRL DMIHER, wardha) and force will be applied along the long axis of roots on the canal with the velocity of 1 mm/min until fracture occurs. The force upon the sample breaking will be recorded in Newton.\n\nThe sample size was calculated using the following formula using mean difference\n\nPrimary variable:- fravture resistance in newton\n\nFracture resistance in cention N group = 734.10 (As per Reference article)\n\nFracture resistance in RMGIC group = 491.60 (As per Reference article)\n\nmean Difference = 242.5 (As per Reference article)\n\nstd. dev = 170.04 (As per Reference article)\n\nAs per reference articles.\n\nTotal samples required = 11 per group.\n\nAll the results will be calculated using SPSS version 27 software. Data for outcome variables will be tested for normality using kalmogorov-smirnov. The comparative analysis over the outcome fracture resistance between three groups (Cention-N, RMGIC, short fiber reinforced flowable composite) will be evaluated on the measurement of fracture resistance for finding significant difference on mean. ANOVA will be used to find the significant difference on mean between 3 groups. Tukey test will be used for comparative evaluation of measurement in between 2 groups pairwise. P-value ≤ 0.05 will be considered as significant at 5% level of significance and 95% confidence of interval.\n\nAdvancements in dental materials will help broaden the perspective to have a holistic approach on using an intraorifce barrier thereby leading to increased strength of teeth which have undergone endodontic treatment. The outcome of this study will help clinicians to choose the best and most efficient filling materials that will increase the fracture resistance of endodontically treated teeth when used as an intraorifice barrier.\n\nNot started yet.\n\nEthical approval was received from the ethical committee of Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha (IEC reference number- DMIHER (DU)/IEC/2023/580) on 06/02/2023.\n\nWritten informed consent will be taken from patients who undergo extraction regarding the use of extracted teeth for the study purpose.\n\n\nDiscussion\n\nRoot canal therapy has enabled dentistry to save teeth that would have been extracted without hesitation just a few decades ago. Endodontically treated teeth, on the other hand, are presumed to be more prone to fracture than vital teeth. After the completion of endodontic treatment, restoration and protection of the remaining tooth structure is of immence importancey.6 Procedures performed after endodontic treatment has gotten more attention, as has their impact on the prognosis of non-vital teeth. But there is no significant difference in moisture content found between endodontically treated teeth and vital teeth.7 The loss of tooth structure is most important in determining the prognosis of the endodontically treated teeth and reinforcing it with an adequate post endodontic restoration along with and intraorifice barrier of high strength and elastic modulus similar to that of dentin is of crucial importance.1 Several studies have shown that force applied along the long axis of the tooth transmits the force uniformly and henceforth in the present study also the force be applied vertically along the long axis of the tooth.8 The materials to be used in the study have properties like high strength and modulus of elasticity near to that of dentin and therefore they will be compared as intraorifice barrier materials in this study. RMGIC (resin modified GIC) was introduced in the late 1980s and contains some methacrylate components found in resin composites. Tselnik et al. reported superior performance as an acceptable coronal seal due to the better performance of RMGIC explained by water sorption by the material, which results in setting expansion and thus a better seal is achieved. It does not require dentin pretreatment and can adhere to it. Another useful property of “RMGIC” is fluoride release. Resin modified GIC has a high flexural strength and modulus of elasticity, and modulus of elasticity values similar to dentin, so the material can withstand a lot of stress before transmitting the load to the root…9–11 Cention N has modulus of elasticity 13 Gpa. It also has patented isofiller which acts as shrinkage stress reliver thus, it helps to relives polymerization shrinkage. It also bonds to tooth structure micromechanically. Isofiller that leads to increased microhardness because filler particles are of nanoparticle size. It helps to withstand stresses and strains of the oral cavity. It can also be placed conservatively thus, reinforcing the remaining tooth structure.12,13 In 2019, the latest type of flowable short fiber reinforced composite (everX Flow, GC, Tokyo, Japan) was launched globally. Several invitro studies have revealed that some SFRCs outperform conventional composites in terms of mechanical and physical performance. Short fibres incorporated in the matrix have significantly improved the material’s ability to resist crack propagation and also lowering the stress intensity at the tip of the crack and it’s propogation in an unstable manner and increase infracture toughness. Many of the properties of fiber-reinforced composites are dependent on microstructural parameters such as fiber diameter, fiber length, fiber orientation, fiber loading, and adhesion of fibers to the polymer matrix.14,15", "appendix": "Data availability\n\nNo data is associated with this article.\n\n\nReferences\n\nDeshpande SR, Gaddalay SL, Damade YN, et al.: Reinforcing the cervical dentin with bonded materials to improve fracture resistance of endodontically treated roots. J. Conserv. Dent. 2022; 25(2): 179–184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChauhan P, Garg A, Mittal R, et al.: A comparative evaluation of fracture resistance of endodontically treated teeth using four different intraorifice barriers: An in vitro study. J. Conserv. Dent. 2019; 22(5): 420–424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoghanizad N, Jones JJ: Evaluation of coronal microleakage after endodontic treatment. J. Endod. 1996 Sep; 22(9): 471–473. Publisher Full Text\n\nYasa E, Arslan H, Yasa B, et al.: The force required to fracture endodontically roots restored with various materials as intra-orifice barriers. Niger. J. Clin. Pract. 2017 Oct; 20(10): 1237–1241. PubMed Abstract \| Publisher Full Text\n\nTortini D, Colombo M, Gagliani M: Apical crown technique to model canal roots. A review of the literature. Minerva Stomatol. 2007 Sep; 56(9): 445–459. PubMed Abstract\n\nAboobaker S, Nair BG, Gopal R, et al.: Effect of Intra-Orifice Barriers on the Fracture Resistance of Endodontically Treated Teeth – An Ex-Vivo Study. J. Clin. Diagn. Res. 2015 Feb; 9(2): ZC17–ZC20. PubMed Abstract \| Publisher Full Text\n\nPapa J, Cain C, Messer HH: Moisture content of vital vs endodontically treated teeth. Endod. Dent. Traumatol. 1994 Apr [cited 2023 Apr 7]; 10(2): 91–93. Publisher Full Text Reference Source\n\nDias de Souza GM, Pereira GDS, Dias CTS, et al.: Fracture resistance of premolars with bonded class II amalgams. Oper. Dent. 2002; 27(4): 349–353. PubMed Abstract\n\nTselnik M, Baumgartner JC, Marshall JG: Bacterial leakage with mineral trioxide aggregate or a resin-modified glass ionomer used as a coronal barrier. J. Endod. 2004 Nov; 30(11): 782–784. PubMed Abstract \| Publisher Full Text\n\nMehta S, Ramugade M, Abrar S, et al.: Evaluation of coronal microleakage of intra-orifice barrier materials in endodontically treated teeth: A systematic review. J. Conserv. Dent. 2022; 25(6): 588–595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams C, Loushine RJ, Weller RN, et al.: A comparison of cohesive strength and stiffness of Resilon and gutta-percha. J. Endod. 2006 Jun; 32(6): 553–555. PubMed Abstract \| Publisher Full Text\n\nChowdhury D, Guha C, Desai P: Comparative Evaluation of Fracture Resistance of Dental Amalgam, Z350 Composite Resin and Cention-N Restoration In Class II Cavity.2019 Jan 25.\n\nJain: Comparative evaluation of three different intraorifice barrier on fracture resistance of endodontically treated teeth: an in vitro study. [cited 2023 Apr 7]. Reference Source\n\nLassila L, Keulemans F, Vallittu PK, et al.: Characterization of restorative short-fiber reinforced dental composites. Dent. Mater. J. 2020; 39(6): 992–999. PubMed Abstract \| Publisher Full Text\n\nVallittu PK: High-aspect ratio fillers: fiber-reinforced composites and their anisotropic properties. Dent. Mater. 2015 Jan; 31(1): 1–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "240458", "date": "20 Feb 2024", "name": "Pallav Mahesh Patni", "expertise": [ "Reviewer Expertise Endodontics", "Dentistry", "Conservative 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\nAbstract is well written and concise. Introduction is clear and to the point. Materials and method are well elaborated and descriptive. Results and Discussion are well explained. Overall, the manuscript contains several sections that could be clarified for better readability and coherence. Additionally, there are some minor errors in grammar, punctuation, and formatting throughout the text that should be addressed.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11752", "date": "11 Jun 2024", "name": "Namrata Jidewar", "role": "Author Response", "response": "Respected Sir, Thank you so much Sir. Regards, Dr. Namrata Jidewar" } ] }, { "id": "271206", "date": "25 May 2024", "name": "Girija S. Sajjan", "expertise": [ "Reviewer Expertise Micro-endodontics", "Guded endodontics", "Regeneration", "aesthetic dentistry", "adhecive 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\n1- Obturation will be performed using the single cone technique with corresponding gutta-percha points and diadent dia-proseal sealer. As most of the premolar pulp space will be not circular in cross-section, warm vertical compaction would be better. 2- All specimens will be prepared by removing coronal 3 mm of gutta-percha with a spoon excavator heated on a Bunsen burner. The removal can be done using TouchNheat equipment with a tip or GG drill would be for better for gp removal rather than a spoon excavator 3- All specimens will be stored at 37°C and at 100% humidity for 1 week in an incubator. Thermal and mechanical loading of the specimen will simulate the oral condition and are indicated.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11751", "date": "26 Jul 2024", "name": "Namrata Jidewar", "role": "Author Response", "response": "Respected ma'am Thank you so much for your valuable suggestions. I have incorporated them in my manuscript to the best of my knowledge and updated the same. Thank You, Regards, Dr. Namrata Jidewar" } ] } ]	1	https://f1000research.com/articles/13-49
https://f1000research.com/articles/12-1452/v1	09 Nov 23	{ "type": "Research Article", "title": "In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV-2 S-glycoprotein and the ACE2 receptor", "authors": [ "Dania Hussein", "Abdullah Almatrafi", "Mohammed Gomaa", "AlAnood Alhowsawi", "Sarah Almustafa", "Hadi Alsaihaty", "Manar Alghamdi", "Abdullah Almatrafi", "Mohammed Gomaa", "AlAnood Alhowsawi", "Sarah Almustafa", "Hadi Alsaihaty", "Manar Alghamdi" ], "abstract": "Background: The protein interaction between the viral surface S-glycoprotein and the host angiotensin converting enzyme-2 receptor (ACE2) is key to the virulent nature of SARS-CoV-2. The potential role that effective drug repurposing strategies may have to help stem the impact of future outbreaks has been brought to light in the recent COVID-19 pandemic. This study outlines a comprehensive approach towards in-silico drug discovery which aims to identify hit agents that can be suitably translated into a clinical setting. Methods: We use two different computational platforms to analyze the viral S-glycoprotein in its bound conformational state to the ACE2 receptor. We employed a comprehensive screening approach to shortlist compounds capable of binding to the viral target interface and corroborated these findings using both Schrödinger’s Glide and AutoDock Vina. Molecular dynamic simulation studies further verified the stability of the interaction at the viral-host protein interface. Results: Lymecycline, pentagalloylglucose, polydatin, and hexoprenaline were identified as prime candidates for further studies given the robust and stable nature of their interaction at the viral-host interface and relevance for clinical testing. These agents were shown in a 100-nanosecond simulation trajectory to favorably disrupt key binding interactions at the viral-host interface and may potentially inhibit viral entry into host cells. In all hit molecules it was observed that inhibiting the interaction with the following key viral binding residues: Lys17, Gly496, Tyr 505, and key host residues: His34, Asp38, Lys353, played a critical role toward the inhibition of the viral-host protein interaction. Conclusions: Our study is unique in its comprehensive approach to identify agents that can bind to the S-glycoprotein-ACE2 interface using multiple computational platforms. Among the hit compounds shortlisted in this study, both lymecycline and hexoprenaline may be considered as candidates for preliminarily clinical studies to assess their therapeutic potential in the management of COVID-19 infections.", "keywords": [ "protein interaction", "Sars-CoV-2", "S-glycoprotein", "Angiotensin converting enzyme-2", "molecular dynamics", "drug repurposing." ], "content": "Introduction\n\nThe novel corona virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had an unprecedented impact worldwide. It is the viral entity responsible for the Coronavirus disease 2019 (COVID-19) pandemic, which has reaped significant detrimental effects upon societies and economies worldwide. Up until present day, efforts remain ongoing towards effective infection management and control.1\n\nThe SARS-CoV-2 virus is an enveloped single stranded RNA virus.2 The entire viral genome has been sequenced and found to comprise of 29,881 bp encoding 9860 amino acids (GenBank no. MN908947).2 The viral RNA codes for both structural and non-structural proteins, most notable of which are the structural S-glycoproteins or ‘spike proteins’- key to viral-host attachment and infection.2 The S-glycoproteins are responsible for binding to the angiotensin converting enzyme 2 (ACE2) host cell receptor and initiating viral entry into the host cell.3 Both SARS-CoV and SARS-CoV-2 bind to the host ACE2 receptor to trigger viral entry and infection, however the binding affinity of the SARS-CoV-2 S-glycoprotein to ACE2 is shown to be over 20 times greater than that of SARS-CoV S-glycoprotein and ACE2 binding interaction.2,4\n\nThe viral S-glycoprotein is a trimeric protein with a characteristic ‘stalk and halo’ like appearance.2 Its peptide chain, composed of a total of 1237 amino acids, includes the S1 (aa residues 14–685) and S2 subunits (aa residues 686–1273), which have been characterized and found to be critical for viral attachment and membrane fusion.5,6 In these regions, both the critical receptor binding domain (aa residues 319–541), as well as the fusion peptide domain (aa residues 788–806), were identified and characterized.4,7\n\nWhile vaccination efforts have greatly stemmed the spread of the infection, the main drawback of long-lasting vaccine efficacy appears to be the growing incidence of variants.8 Managing the disease with currently available and approved therapeutics, remains an essential approach for treatment. However, there appears to be is no consensus on effective management strategies yet.9,10 Drug repurposing is an approach to identify novel indications and uses for approved medications. In the era of COVID-19 drug repurposing has been shown to be an essential path towards identifying potentially effective therapies for disease management.11,12 Strategies towards this include in silico drug discovery tools and virtual screening, which have proven to be most valuable in identifying potential drugs that can be repurposed towards a new target.11,13 Utilizing computational modelling platforms to visualize and identify ligand-target interactions is a powerful and efficient approach towards successful drug development.14\n\nThis study aims to apply a comprehensive virtual screening approach using multiple platforms to identify drugs and natural products that may potentially be repurposed to inhibit the binding interaction between the SARS-CoV-2 S-glycoprotein and the host ACE2 receptor. Furthermore, the interaction profile of all hit compounds is verified by molecular dynamic simulation studies to provide a detailed insight towards the stability and nature of the binding interaction at the viral-host interface.\n\n\nMethods\n\nThe molecular modelling software Maestro by Schrödinger15 and AutoDock Vina,16 were both used for virtual screening and molecular dynamics simulation studies. The desktop workstation was equipped with Intel® Core™ i7-10700F Processor, Linux Ubuntu 22.10 operating system and a RTX 5000 graphics card.\n\nThe protein crystal structures were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (RRID:SCR_012820). For virtual screening and molecular dynamic simulations, the structure of the SARS-CoV-2 S-glycoprotein receptor binding domain (S-RBD) bound to the ACE2 receptor interface was used (PDB code: 6M0J).\n\nA ligand library was compiled comprised of Food and Drug Administration (FDA) approved and worldwide approved drugs as well as approved nutraceuticals and natural compounds with verified in vivo efficacy. A total of 1100 FDA approved drugs were retrieved in SDF format from DrugBank database (RRID:SCR_002700),17 along with a library of 3440 worldwide approved medications and 74 approved nutraceuticals. Furthermore, 1537 natural compounds with verified physiological effects in vivo, were retrieved from Zinc database (RRID:SCR_006082).18 Table 1 summarizes the ligand categories of the compiled virtual library.\n\nProtein preparation\n\nAll crystal structures were prepared using the Schrödinger Maestro’s (RRID:SCR_016748) protein preparation wizard tool. Structure preparation and minimization was done at a pH 7.4 with corrected ionization states, polar hydrogens were added, and non-essential water molecules were removed. The entire structure was minimized and optimized with the OPLS3 force field and the default value for the RMSD of 0.30 Å was used for non-hydrogen atoms.\n\nLigand library preparation\n\nDownloaded SDF structures were prepared for docking studies using the maestro Ligprep tool (RRID:SCR_016748). Structures were converted into 3D maestro format, and ionization states and chirality were optimized at physiological pH (7.4) using OPLS3 force field. The final 3D conformations were utilized for virtual screening.\n\nBinding pocket determination and docking studies\n\nIn Schrödinger’s Maestro (RRID:SCR_016748) the binding pocket was identified using Schrödinger’s Sitemap, a single binding pocket between the interface of the RBD and ACE2 binding region (PBD 6M0J) was used. The selected binding pockets were verified by ensuring the presence of essential binding residues identified in previous studies.6 The site score value was within the range of 1-1.1 for each of the binding pockets indicating high accessibility and druggability of the selected binding pocket. The selected binding pockets were used to generate a docking grid using Maestro’s Glide module for docking studies. The receptor grids were generated using the prepared proteins, with the docking grids centered on the identified receptor binding pocket for each protein. A receptor grid was generated using a 1.00 van der Waals (vdw) radius scaling factor and 0.25 partial charge cut-off. The binding sites were enclosed in a grid box of 20 Å3 without constraints and using default parameters. Docking was repeated and verified using three screening settings. All compounds were screened under a high-throughput docking setting and the top 200 compounds with the highest binding scores where then selected for standard precision docking, of these verified hits the top 80 compounds where further verified using extra precision (XP) docking settings. The ligands were docked using the extra precision mode (XP) without using any constraints and a 0.80 van der Waals (vdw) radius scaling factor and 0.15 partial charge cut-off. Induced fit docking was carried out with flexibility of the residues of the pocket near to the ligand.\n\nGlideScore implemented in Glide (RRID:SCR_016748), was used to estimate binding affinity and rank ligands. The XP Pose Rank was used to select the best-docked pose for each ligand. The final list of thrice verified compounds was then analyzed in detail based on binding scores and a detailed study of all binding interactions.\n\nMolecular dynamics simulation studies\n\nMolecular dynamic (MD) simulation studies were carried out using the Desmond Module on Schrödinger’s Maestro platform (RRID:SCR_016748). The protein preparation wizard was used to minimize the hit protein-ligand complex and the simulation environment was built using the system builder application of Desmond. A water based solvent system: TIP3P was employed to generate the simulation environment contained within an orthorhombic simulation box with 10 Å buffer parameter from the protein surface. The system was neutralized and isotonic conditions attained via the addition of counter ions and 0.15 M NaCl. All MD simulations were conducted at a temperature of 300 K and a pressure of 1.013 bar. A simulation period of 100 nanoseconds was run for each of the hit ligand-protein complexes. Analysis calculations were subsequently run and results presented using the simulation interaction diagram tool of Desmond.\n\nDocking calculations were carried out using the AutoDock vina software version 1.1.2 (RRID:SCR_011958).16 All hydrogens were added to the ligand PDB file and Gasteiger charges were computed and all the torsion angles of the ligand were defined using the autodock-tools program. A grid box generated with the following dimensions: 36×24×-4 Å, with a grid spacing of 1 Å was used. The Lamarckian genetic algorithm was used as a search method with a total of 30 runs (maximum of 20 000 000 energy evaluations; 27 000 generations; initial populations of 150 conformers). The binding affinity calculation in AutoDock vina together with analysis of binding interactions were used to select hits for molecular dynamic simulation studies.\n\n\nResults\n\nPreliminary docking studies to identify ligands that interact favorably with the receptor binding domain of the spike protein, involved screening a total of over 7000 approved medications and natural compounds (see Table 1). Initial docking studies were carried out on the S-RBD and ACE2 interface using Schrödinger Glide and Autodock Vina to identify hits that would prevent/disrupt the crucial protein-protein interaction. The results from both programs were analyzed and compared. The compounds that showed the most favorable binding profile and the best binding scores were shortlisted in Table 1.29,30\n\nFrom the initial hits 13 were identified that showed consistent good binding scores and binding poses. The compounds shortlisted bound to the protein-protein interface, and established interactions with key residues namely, His34, Asp38, and Lys353 from the ACE-2 binding region and Lys417, Gly496 and Tyr505 from the S-RBD. Hexoprenaline and tricocin, being highly flexible molecules and n-acetylglucosamine being a relatively small molecule were able to burrow deeply within the protein interface, while maintaining interactions with the above-mentioned key residues (Figure 1). The hit compounds all expressed high binding affinity scores using both platforms; ranging from -7.5 — -12.4 (Glide scoring), and -4.8 — -8.9 (Autodock vina scoring).\n\nACE-2 residues with carbon in green and S-glycoprotein residues with carbon in brown. The hit compounds are represented in yellow with hexoprenaline, n-acetyglucosamine, and tricocin highlighted in magenta.\n\nLymecycline, as an example, established key interactions via its side chain groups (Figure 2). The carboxylate formed a crossbridge at the interface; with strong charge assisted H bond between the Asp38 and Lys353 sidechains from ACE-2 and the Gly496 sidechain from the spike protein. The interaction was further strengthened by the ammonium ion of the terminal amino acid forming a charge assisted H bond with both the His34 backbone (ACE2) and Gly496 sidechain (S-RBD). A water mediated H bond for this ammonium ion with the Tyr453 sidechain of the S-RBD was also observed. Potential H bonds were also noted between the sidechain amide oxygen and Lys417, as well as the ring’s tertiary amine with Tyr505 from the spike protein. An increase of selectivity for lymecycline is expected owing to the presence of two opposite electrostatic interactions with both chains. The first a H bond between the sidechain amide and His34 sidechain (ACE-2) and the second is a H-π interaction between the central aliphatic ammonium and Tyr453 aromatic ring (S-RBD).\n\nACE-2 residues with carbon in green and S-glycoprotein residues with carbon in brown. Potential electrostatic interactions are represented as red dotted lines and distances are in Angstrom.\n\nThe top hit molecules were selected based on a detailed visual analysis of the interaction profile of each compound with the target proteins. A hit was identified as any compound which had a favorable binding score and profile. Compounds were shortlisted based on reproducibility of the results using two different docking platforms (Glide and AutoDock Vina). 13 hit ligands were selected for molecular dynamic simulation studies (Table 2). Investigation of the optimal 2D and 3D docked positions reveled that each of the hit ligands form interactions with key residues of the binding pocket. Root mean square deviation (RMSD) plot analysis was used to measure the average displacement of atoms with respect to a reference frame. RMDS analysis revealed a stable binding profile for the top 7 ligands as shown in Figure 3 (RMSD fluctuation range of 2-4 Å). Of the 7 hit ligands, 3 exhibited a highly stable and robust binding profile within the interface binding pocket, these include: lymecycline, pentagalloylglucose and polydatin. The interaction profile for each individual ligand is depicted in Figures 4-6 (for MD results for the remaining 4 ligands see extended data). As the S-RBD-ACE2 interface structure was used for simulations, all interactions of the ligand with key binding residues of both chain A of the ACE2 binding domain and chain E of the S-RBD were considered as significant.\n\nThe green graph shows fluctuations in the protein backbone from the initial reference point while the red shows the ligand fluctuations. The RMSD profile of the ligand is with respect to its initial fit to the protein binding pocket indicates that all ligands did not fluctuate beyond a 2-4 Å range.\n\n(A) Interaction of lymecycline with residues in each trajectory frame. The depth of color indicating the higher the interaction with contact residues; (B) The protein-ligand contacts showing the binding interactions fraction; (C) Lymecycline interactions with the protein residues during MD simulation. Interactions shown are occurring more than 30% during the simulation time. A: chain A of the ACE2 binding domain, E: chain E of the S-glycoprotein receptor binding domain.\n\n(A) Interaction of Pentagalloylglucose with residues in each trajectory frame. The depth of color indicating the higher the interaction with contact residues; (B) The protein-ligand contacts showing the bonding interactions fraction; (C) Pentagalloylglucose interaction with the protein residues during MD simulation. Interactions shown are occurring more than 30% during the simulation time.\n\n(A) Interaction of polydatin with residues in each trajectory frame. The depth of color indicating the higher the interaction with contact residues; (B) The protein-ligand contacts showing the bonding interactions fraction; (C) Polydatin interaction with the protein residues during MD simulation. Interactions shown are occurring more than 30% during the simulation time.\n\nLymecycline shows an exceptional binding profile with key binding residues of the binding pocket as shown in Figure 4. Key binding residues of the S-RBD Lys417 and Gly496 form a H bonding interaction with the lymecycline, furthermore it exhibited water bridge interactions with Tyr 505 and Ser494. While at the interface lymecycline was also shown to form interactions with key binding residues of the ACE2 binding domain including a strong H bond with His34 and mixed interactions (ionic and hydrogen bonds) with residues Asp38 and Lys353. All significant interactions are represented in Figures 4 A and B, which highlight residues with the strongest ligand interactions that are stable over the entire simulation period. Figure 4 C depicts all significant interactions displayed by the ligand and interacting residues occurring for over 30% of the simulation period. Of significance Lys417 and Gly496 are bound to lymecycline over 60% of the simulation period.\n\nMD simulation results for pentagalloylglucose in Figure 5 show significant interactions with key residues of the binding pocket. Key binding residues of the S-RBD: Lys417 and Gly496, form H bonds, and Lys417 was also shown to form a water bridge interaction with the ligand. Additionally, a stable hydrophobic interaction with residue Tyr505 was observed. Strong interactions with key residues of the ACE2 binding domain were also observed in the simulation period, notably H bonds and water bridges with residues Asp38 and Arg393. All significant interactions are represented in Figures 5A‐C. Of significance Lys417 interacts with pentagalloylglucose approximately 85% of the simulation period.\n\nPolydatin exhibits several stable interactions throughout the 100 nanosecond simulation period as depicted in Figure 6. Polydatin binds with key residues of the S-RBD Lys417 and Tyr505. A mixed binding profile is observed including H bond interactions, water bridges and for Lys417 hydrophobic interactions. Polydatin was also shown to form interactions with key residues of the ACE2 binding domain including a strong hydrogen bond with His37 and mixed H bond and water bridge interaction with residue Asp30. All significant interactions are represented in Figure 6.\n\n\nDiscussion\n\nOver the last two decades, different docking tools and programs have been developed that use different algorithms in which the conformation of the ligand is extensively evaluated in a binding pocket until an energy minimum is reached. Most programs treat the ligand as a flexible component and the receptor as rigid while others treat both interacting components (ligand and receptor) as flexible.16 Such programs not only differ in the type of docking, but also in their ligand placement strategies.19 In this study, we selected Schrödinger’s Maestro and Autodock vina in order to assess the docking accuracy and mode of binding. Autodock vina was used to perform rigid docking while maestro was used for induced fit (flexible) docking. Autodock uses the genetic algorithm while maestro uses systematic search techniques for ligand placement.\n\nThis study was a comprehensive in silico investigation of a key target of the SARS-CoV-2 virus; the surface S-glycoprotein or spike protein. The interaction between the S-glycoprotein and target ACE2 host receptor is essential to the virulent nature of the virus. Agents that can disrupt this binding interaction may potentially inhibit viral entry into the host cell and infection. Structures selected for this study were all solved by x-ray crystallography or EM with a minimum accepted resolution of 2.45 Angstrom. For each of the target structures, the selected binding pockets from Schrödinger’s Sitemap were verified by ensuring the presence of essential binding residues identified in previous studies (Table 2). A detailed study of the viral host interface depicted in the 6M0J crystal structure revealed the key residues involved in strong charge assisted H bonds, ionic bonds, and strong H bonds, judged by the bond length, charge, and orientation (Table 3). Binding to these residues is necessary to disrupt the natural interaction between the two proteins in favor of the hit ligand.\n\nIn our study, a comprehensive screening library of over 7000 compounds was compiled, comprised of both FDA and worldwide approved drugs and nutraceuticals in addition to all natural products with established in vivo activity (Zinc)18 (Drugbank).17 In order to ensure robust and reproducible results virtual screening was preformed using two different platforms: Glide15 and Autodock Vina.16 The thorough protocol employed within this study ensures that the hit ligands identified had verified binding profiles. Further molecular dynamic simulation studies were vital towards verifying and visualizing the nature of the binding interaction of the hit ligands within the binding interface. Hits, that were selected for MD simulation studies, were determined to be the most likely to disrupt the strongest interactions between the viral and host proteins (Table 2).\n\nMD simulation studies identified the following hit ligands: lymecycline, hexoprenaline, pentagalloylglucose, polydatin, tricrocin, setmelanotide and forsythiaside. All hits expressed a stable interaction profile as indicated by RMSD below 4 Å for both proteins and ligand position. It is important to highlight that all compounds were shortlisted from initial screening results based not only on their binding profile but also their suitability to be translated towards a clinical setting. Several drugs used in the management of COVID-19 have had detrimental effects owing to the adverse drug reaction profile of the employed therapeutic. The pathogenesis of COVID-19 culminates in several immune and cardiovascular manifestations ranging from hypercoagulability to kidney failure, as of such agents that are not appropriately selected may cause more harm than benefit within the overall scope of disease management.20 Four hits were selected including lymecycline, pentagalloylglucose, polydatin, and hexoprenaline, which expressed very good RMSD profiles, indicating that the ligand remained bound in a stable manner within the binding pocket throughout the entirety of the simulation period. Other shortlisted ligands such as setmelanotide, and forsythiaside appear to have fluctuated with respect to their initial position relative to the protein backbone at some point within the simulation period although, the range of the fluctuation did not exceed beyond the range of 2-4 Å. The three final hits; lymecycline, pentagalloylglocose and polydatin, maintain strong bonds with residues within the binding pocket and in some cases vital binding residues throughout the entire simulation period.\n\nLymecycline, a broad-spectrum second-generation tetracycline antibiotic commonly used in the management of acne, gynecological and respiratory tract infections, was shown to exhibit stable binding with key binding residues of both the spike and ACE2 RBD. Lymecycline maintained its interaction with its side chain throughout the simulation period. The stable charge assisted H bond with Lys 353 from ACE-2 and Gly 496 from spike was conserved during the dynamic simulation which suggests that these interactions are more energetically favored over the initial H bond between the two mentioned residues. Other crucial interactions are shown with residues; Glu 37 and Asp 38 from ACE-2 and Tyr 505 from the S-RBD. These residues were bound via a H bond in the original protein-protein interaction. Lymecycline has been reported in previous studies to bind to additional viral targets in the SARS-CoV-2 virus, namely the main protease (Mpro).21 Like many members of its class it expresses anti-inflammatory properties,22 this makes it a particularly attractive subject that can be further investigated for repurposing studies in the treatment of SARS-CoV-2.\n\nPentagalloylglucose is a polyphenolic compound that has been shown in in vitro and in vivo studies to have anti-viral effects against the hepatitis C virus.23 A number of recent in vitro studies have shown a dose dependent effect in inhibiting viral spike association with the host ACE2 receptor.24 Our study reveals the detailed nature of the binding interaction of pentagalloylglucose at the S-RBD-ACE2 binding interface. Key residues Lys417, Gly496 and Tyr505 of the viral spike protein and residues Asp38 and Arg393 of the ACE2 binding domain, form a stable binding interaction with pentagalloylglucose. Interference with viral-host binding at these key residues is very likely to be vital for the inhibition of viral engagement with the host ACE2 receptor and subsequent cell entry.\n\nPolydatin, another polyphenolic compound, is a glycoside precursor of resveratrol. In vitro studies have shown its potential to inhibit viral spike protein binding with the ACE2 receptor.25 The findings of this study corroborate our own where similar binding scores were reported. Furthermore, our studies identify strong interactions of the key binding residues Lys417 of the S-RBD and Asp30 and Glu37 of the ACE2 binding region with polydatin. It appears that interfering with the binding of the viral Lys417 residue is a key inhibitory pattern detected in a number of docking profiles supported by in vitro findings.24,25\n\nHexoprenaline, a β2 adrenoceptor agonist, is mentioned as a drug of interest although it was not identified from the top three- hits that showed the most stable interactions during MD simulations. It is for the first time reported to exhibit a favorable binding profile with a number of key binding residues of the S-glycoprotein RBD as well as the ACE2 binding domain. Hexoprenaline was shown to bind key residues and to interrupt key interactions. One of its side-chain nitrogen atoms formed two charge assisted H bonds with the two initially bound residues Glu35 from ACE-2 and Gln493 from S-RBD. The other chain nitrogen formed a charge assisted H bond with Glu37 (see extended data for MD results of remaining shortlisted compounds). The stability of these critical bonds during dynamics was slightly lower than those noted for lymecycline during the simulation period. Yet, considering its role as a bronchodilator and its pharmacological profile it may be considered a good candidate for drug repurposing in the management of COVID-19 infection where there is a high incidence of respiratory distress. However, caution must be taken, considering its potential nonselective activity on β1 receptors which may result in unwanted cardiovascular effects.26\n\nIn this study lymecycline, pentagalloylglucose, and polydantin were identified as potential inhibitors of the S-RBD-ACE2 binding interface. Hexoprenaline could be also considered as a promising hit, due to its favorable docking and dynamic profile and taking into account its relevance and suitability for clinical testing. Of the nutraceuticals, forsythiaside also appears promising in its ability to potentially disrupt key binding interactions at the viral-host interface and is a prime candidate for further in vitro and in vivo studies. From a clinical perspective, both lymecycline and hexoprenaline may be considered as possible candidates for preliminarily clinical studies to assess their therapeutic potential in the management of COVID-19 infections.\n\n\nConclusions\n\nVirtual screening is a highly attractive approach to identify potential compounds with therapeutic efficacy against target proteins, while efficient, it is not without its limitations. There have been a large number of docking studies published in the literature that have identified agents that may potentially be repurposed to inhibit SARS-CoV-2 targets. Our study is unique in its comprehensive approach to identify agents that can bind to the viral S-glycoprotein-ACE2 binding interface using multiple platforms. Molecular dynamic simulation studies were essential to identify a consistent binding pattern that appears to be common in the most effective agents that have the potential to inhibit the S-glycoprotein-ACE2 interaction. The agents identified in this study were additionally shortlisted for their suitability to be translated to a clinical COVID-19 setting by understanding their toxicity profile and identifying agents with verified anti-inflammatory and anti-viral capacity. The hit compounds identified are prime agents for further in vitro and clinical investigations to verify their efficacy in the potential treatment of COVID-19.\n\n\nAuthor contributions\n\nD.H. designed the research project. D.H., M.G., A.A., and A.H. conducted the computational studies. D.H. and M.G. analyzed the results. S.M., H.A., M.A., helped draft the manuscript. All authors were involved in writing, editing and revision of the manuscript.", "appendix": "Data availability\n\nSource data include the protein crystal structures which were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (RRID:SCR_012820). http://www.rcsb.org/#Category-welcome . The PDB accession code for the protein crystal structure of the S-glycoprotein bound to the ACE-2 receptor is 6M0J, and the pdb sturcture file can be accessed here: https://www.rcsb.org/structure/6m0j.\n\nAdditional source data include the ligand libraries which were retrieved from online databases: DrugBank database (RRID:SCR_002700) found here: http://www.drugbank.ca/. Annual account subscription and access to ligand libraries was kindly provided for the purpose of academic research. Freely available ligand libraries were downloaded from the open access Zinc database (RRID:SCR_006082) found here http://blaster.docking.org/zinc/. See Table sd1 below and/or in Table 1 in the main text for complete details.\n\nFigshare: RAW DATA FILE: In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV -2 S-glycoprotein and the ACE2 receptor. https://doi.org/10.6084/m9.figshare.22257352.v5. 30\n\nThis project contains the following underlying data:\n\nSimulation results: final trajectory frames of the hit compounds bound to the protein interface:\n\n- ligprep_DrugBannkApprovedNOMETAL28072021_maegz_1967.pdb\n\n- ligprep_DrugBannkApprovedNOMETAL28072021_maegz_2048.pdb\n\n- ligprep_DrugBannkApprovedNOMETAL28072021_maegz_135.pdb\n\n- ligprep_DrugBannkApprovedNOMETAL28072021_maegz_7.pdb\n\n- ligprep_DrugBannkApprovedNOMETAL28072021_maegz_654.pdb\n\n- ligprep_WorldappNOTFDAZinc_maegz_3777.pdb\n\n- ligprep_DrugBannkNaturaceuticalNOMETAL28072021_2_maegz_83.pdb\n\n- ligprep_DrugBannkNaturaceuticalNOMETAL28072021_2_maegz_5.pdb\n\n- ligprep_DrugBannkNaturaceuticalNOMETAL28072021_2_maegz_27.pdb\n\n- ligprep_NaturalINvivoZinc_NOMETAL_maegz_1403.pdb\n\n- ligprep_NaturalINvivoZinc_NOMETAL_maegz_1472.pdb\n\n- ligprep_WorldappNOTFDAZinc_maegz_3450.pdb\n\n- ligprep_WorldappNOTFDAZinc_maegz_40.pdb\n\nProtein structure:\n\n- spikeACE2RBDinterface_protein.pdb\n\nLigand libraries derived from open-source databases:\n\n- FDAapproved medications.sdf\n\n- world-not-fdaApproved medication.sdf\n\n- NaturalcompoundsLibrary.sdf\n\nExtended Data: In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV -2 S-glycoprotein and the ACE2 receptor. https://doi.org/10.6084/m9.figshare.22414234.v1. 30\n\nThis project contains the following extended data:\n\n- Extended Data Insilico Drug repurposing SglycoproteinACE2.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors acknowledge the support from the College of Clinical Pharmacy of Imam Abdulrahman Bin Faisal University, Saudi Arabia.\n\n\nReferences\n\nChakraborty I, Maity P: COVID-19 outbreak: Migration, effects on society, global environment and prevention. Sci. Total Environ. 2020; 728: 138882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Liu Q, Guo D: Emerging coronaviruses: Genome structure, replication, and pathogenesis. J. Med. Virol. 2020; 92(4): 418–423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang Y, Yang C, Xu X-f, et al.: Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol. Sin. 2020; 41(9): 1141–1149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWrapp D, Wang N, Corbett KS, et al.: Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367(6483): 1260–1263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann M, Kleine-Weber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2): 271–80.e8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Q, Zhang Y, Wu L, et al.: Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell. 2020; 181(4): 894–904.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalls AC, Park YJ, Tortorici MA, et al.: Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020; 181(2): 281–92.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi JY, Smith DM: SARS-CoV-2 Variants of Concern. Yonsei Med. J. 2021; 62(11): 961–968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeigel JH, Tomashek KM, Dodd LE, et al.: Remdesivir for the Treatment of Covid-19 — Final Report. N. Engl. J. Med. 2020; 383(19): 1813–1826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoman YM, Burela PA, Pasupuleti V, et al.: Ivermectin for the treatment of COVID-19: A systematic review and meta-analysis of randomized controlled trials. Clin. Infect. Dis. 2021.\n\nEweas AF, Alhossary AA, Abdel-Moneim AS: Molecular Docking Reveals Ivermectin and Remdesivir as Potential Repurposed Drugs Against SARS-CoV-2. Front. Microbiol. 2021; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDas NC, Chakraborty P, Bayry J, et al.: In Silico Analyses on the Comparative Potential of Therapeutic Human Monoclonal Antibodies Against Newly Emerged SARS-CoV-2 Variants Bearing Mutant Spike Protein. Front. Immunol. 2022; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTouret F, Gilles M, Barral K, et al.: In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication. Sci. Rep. 2020; 10(1): 13093. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodds MG, Krishna R, Goncalves A, et al.: Model-informed drug repurposing: Viral kinetic modelling to prioritize rational drug combinations for COVID-19. Br. J. Clin. Pharmacol. 2021; 87(9): 3439–3450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriesner RA, Murphy RB, Repasky MP, et al.: Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 2006; 49(21): 6177–6196. PubMed Abstract \| Publisher Full Text\n\nTrott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010; 31(2): 455–461. PubMed Abstract \| Publisher Full Text\n\nWishart DS, Feunang YD, Guo AC, et al.: DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res. 2018; 46(D1): D1074–D1082. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIrwin JJ, Sterling T, Mysinger MM, et al.: ZINC: A Free Tool to Discover Chemistry for Biology. J. Chem. Inf. Model. 2012; 52(7): 1757–1768. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPagadala NS, Syed K, Tuszynski J: Software for molecular docking: a review. Biophys. Rev. 2017; 9(2): 91–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelly M, O'Connor R, Townsend L, et al.: Clinical outcomes and adverse events in patients hospitalised with COVID-19, treated with off-label hydroxychloroquine and azithromycin. Br. J. Clin. Pharmacol. 2021; 87(3): 1150–1154. PubMed Abstract \| Publisher Full Text\n\nWu C, Liu Y, Yang Y, et al.: Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods. Acta Pharm. Sin. B. 2020; 10(5): 766–788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradhan S, Madke B, Kabra P, et al.: Anti-inflammatory and Immunomodulatory Effects of Antibiotics and Their Use in Dermatology. Indian J. Dermatol. 2016; 61(5): 469–481. PubMed Abstract \| Publisher Full Text\n\nBernardi M, Ghaani MR, Bayazeid O: Phenylethanoid glycosides as a possible COVID-19 protease inhibitor: a virtual screening approach. J. Mol. Model. 2021; 27(11): 341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen RH, Yang LJ, Hamdoun S, et al.: 1,2,3,4,6-Pentagalloyl Glucose, a RBD-ACE2 Binding Inhibitor to Prevent SARS-CoV-2 Infection. Front. Pharmacol. 2021; 12: 12. Publisher Full Text\n\nPerrella F, Coppola F, Petrone A, et al.: Interference of Polydatin/Resveratrol in the ACE2:Spike Recognition during COVID-19 Infection. A Focus on Their Potential Mechanism of Action through Computational and Biochemical Assays. Biomolecules. 2021; 11(7): 1048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinder RM, Brogden RN, Speight TM, et al.: Hexoprenaline: a review of its pharmacological properties and therapeutic efficacy with particular reference to asthma. Drugs. 1977; 14(1): 1–28. Publisher Full Text\n\nLan J, Ge J, Yu J, et al.: Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020; 581(7807): 215–220. PubMed Abstract \| Publisher Full Text\n\nVerkhivker GM: Molecular Simulations and Network Modeling Reveal an Allosteric Signaling in the SARS-CoV-2 Spike Proteins. J. Proteome Res. 2020; 19(11): 4587–4608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHussein D, Mansour MS, Al Matrafi A , et al.: Underlying data: In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV -2 S-glycoprotein and the ACE2 receptor. Dataset. figshare. 2023. Publisher Full Text\n\nHussein D: Extended Data: In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV -2 S-glycoprotein and the ACE2 receptor. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "244892", "date": "01 Mar 2024", "name": "Prateek Kumar", "expertise": [ "Reviewer Expertise Bioinformatics", "Molecular Biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview Report: The title of manuscript is interesting but it requires major revisions before getting approved. Authors are requested to incorporate following suggestions:\nMajor revisions:\n1. Validation of docking results is missing, please include the validation of molecular docking. 2. Perform ADMET analysis to check the drug abilities/potential of screened compounds. 3. Perform binding free energy calculations MM/GBSA.\nMinor revisions: 4. Improve the conclusion part in Abstract as well as the final conclusion. It should be more conclusive not broad. 5. Manuscript require thorough checking of Grammer and typo errors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "12019", "date": "26 Jul 2024", "name": "dania hussein", "role": "Author Response", "response": "We thank the esteemed reviewer for his valued input and comments. We have addressed and responded to each comment accordingly: Major revisions: 1. Validation of docking results is missing, please include the validation of molecular docking. We thank the esteemed reviewer for their valuable comments and helpful suggestions. We have added a additional paragraph regarding the protocol validation. Due to the absence of any verified crystal structure of ligands bound to the SARS-Cov-2 receptor binding domain and ACE2 interface we utilized the Site Score tool on Maestro to identify and calculate the region with the highest druggability that encompasses the interactions of interest. The key interactions between the viral host and receptor have been determine and verified and we were keen to identify agents that could disrupt such interactions. Using SiteScore the binding pocket with thus determined and for validation we employed 2 different docking platforms: Glide and AutoDoc Vina. For an additional level of validation supported by experimental evidence ligands with verified activity against the S-glycoprotein-ACE2 interaction were docked using Glide. Biological activity was assessed and IC50 values determined in previous studies, this was compared to the determined binding affinities represented by the docking scores attained using the extra precision docking protocol. The findings provided another tier of validation highlighting a complementary association between the calculated binding affinities and experimental activity of our designated control ligands, this data is available in the Extended Data file. 2. Perform ADMET analysis to check the drug abilities/potential of screened compounds. Thank you for this suggestion ADMET values were derived for the top seven ligands are presented as a table in the manuscript. 3. Perform binding free energy calculations MM/GBSA. Thank you for this valuable suggestion, we have accordingly preformed free energy calculations and added this data to the manuscript. Minor revisions: 4. Improve the conclusion part in Abstract as well as the final conclusion. It should be more conclusive not broad. The abstract conclusion part was rewritten as follows: Our study represents a rational drug discovery approach that utilizes a unique and comprehensive approach to identify agents that can bind to the S-glycoprotein-ACE2 interface using multiple computational platforms. Seven hits were identified: lymecycline, hexoprenaline, pentagalloylglucose, polydatin, tricrocin, setmelanotide and forsythiaside. Among the hit compounds shortlisted in this study, both lymecycline and hexoprenaline were considered to be two significant drug hits in terms of their applicability in COVID-19 clinical settings and therefore are considered primary candidates for translational and clinical studies to assess their therapeutic potential in the management of COVID-19 infections. The conclusion part was rewritten as follows: There have been a large number of docking studies published in the literature that have identified agents that may potentially be repurposed towards inhibiting SARS-CoV-2 targets. However, our study represents a unique and comprehensive approach to repurpose drugs that can bind to the viral S-glycoprotein-ACE2 binding interface using multiple platforms. Two different docking platforms were utilized and binding free energy calculations and molecular dynamic simulation studies were performed to identify a consistent binding pattern that appears to be common in the most effective agents that have the potential to inhibit the S-glycoprotein-ACE2 interaction. Seven drugs were identified as hits including, lymecycline, hexoprenaline, pentagalloylglucose, polydatin, tricrocin, setmelanotide and forsythiaside. The hits identified in this study were additionally shortlisted for their suitability to be translated to a clinical COVID-19 setting by understanding their toxicity profile and identifying agents with verified anti-inflammatory and anti-viral capacity. In our study lymecycline, and hexoprenaline are proposed as prime candidates for further translational, preclinical and clinical investigations for the treatment of COVID-19. 5. Manuscript require thorough checking of Grammar and typo errors. Thank you for this helpful observation, we have accordingly revised the manuscript for mistakes." } ] }, { "id": "260767", "date": "20 May 2024", "name": "Dr Manne Munikumar", "expertise": [ "Reviewer Expertise Molecular Docking", "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\nDear Authors, The manuscript entitled “In silico drug repurposing using molecular docking and dynamics to target the protein interaction between the SARS-CoV-2 S-glycoprotein and the ACE2 receptor.” thoroughly assessed. The manuscript demonstrates a rigorous methodology, employing two different computational platforms for virtual screening, molecular docking, and molecular dynamics simulations. This comprehensive approach enhances the reliability and robustness of the findings. The study addresses an important and timely topic by focusing on the protein interaction between the SARS-CoV-2 S-glycoprotein and the ACE2 receptor, which is crucial for viral entry and infection. The potential for drug repurposing strategies to combat COVID-19 is underscored, highlighting the significance of the research. The manuscript successfully identifies several potential hit compounds with stable interactions at the viral-host interface. The selection of lymecycline, pentagalloylglucose, polydatin, and hexoprenaline for further investigation demonstrates promising results and provides a basis for future experimental studies. The study provides valuable insights into the binding mechanisms of the identified hit compounds, elucidating their interactions with key viral and host residues. This deepens understanding of the molecular basis of SARS-CoV-2 infection and potential therapeutic interventions. The conclusion offers practical implications by suggesting lymecycline and hexoprenaline as candidates for preliminary clinical studies. This highlights the translational potential of the findings and their relevance for the development of COVID-19 therapeutics. Following comments has to be addressed.\nWhile the computational approach is thorough, experimental validation of the findings is lacking. Incorporating in vitro or in vivo studies to confirm the efficacy of the identified compounds would strengthen the manuscript and provide more robust evidence for their potential as therapeutics. The manuscript could benefit from a more comprehensive discussion of the limitations and assumptions of the computational methods employed. Addressing factors such as binding site flexibility, solvent effects, and conformational changes could enhance the interpretation of the results and their relevance to real-world scenarios. The study focuses on a specific set of compounds and interactions, which may limit its generalizability to other drug candidates or viral strains. Discussing the broader implications of the findings and potential applicability to related viruses or drug targets would enrich the manuscript. It would be beneficial for the authors to provide information on the availability of the datasets, software codes, and molecular structures used in the study. Enhancing data accessibility would promote transparency and reproducibility in computational drug discovery research. While the selected compounds show promise in silico, their clinical feasibility and safety profiles need to be thoroughly evaluated. Discussion of potential side effects, pharmacokinetic properties, and drug interactions would provide a more comprehensive assessment of their suitability for clinical trials.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "12018", "date": "26 Jul 2024", "name": "dania hussein", "role": "Author Response", "response": "We thank the esteemed reviewer for his valued input and comments. We have addressed and responded to each comment accordingly: While the computational approach is thorough, experimental validation of the findings is lacking. Incorporating in vitro or in vivo studies to confirm the efficacy of the identified compounds would strengthen the manuscript and provide more robust evidence for their potential as therapeutics. We thank the reviewer for this valuable comment. The current research involves in silico drug repurposing for COVID-19 through identifying potential ligands that bind at the protein-protein interface and inhibits the interaction of SARS-CoV -2 S-glycoprotein and the ACE2 receptor. This represents the first step of a translational research project to shortlist potential binders to be followed by in vitro and in vivo studies and finally clinical testing and repositioning. A further level of validation has been incorporated by considering historical evidence of biological activity of experimental ligands known to disrupt the viral host (S-RBD) and receptor (ACE-2) interaction. We have been able to show that experimental agents with biological activity also express binding affinity using our employed docking protocol. The manuscript could benefit from a more comprehensive discussion of the limitations and assumptions of the computational methods employed. Addressing factors such as binding site flexibility, solvent effects, and conformational changes could enhance the interpretation of the results and their relevance to real-world scenarios. We thank the reviewer for this observation and have included the following paragraph to the discussion section: The current study benefited from a cross-docking protocol that utilized two different programs with two different algorithms to validate the results of the predicted docking poses of potential binders and increase the likelihood of getting satisfying results when proceeding with future laboratory and clinical testing. This was also further supported with a MD simulation for the best hits. MD simulation addresses important factors not included in the docking calculations such as binding site flexibility, solvent effects, and conformational changes, and thereby confirms the stability and the accuracy of the predicted docking conformations and increases the probability of being adopted in a real biological environment. Of interest a number of hit ligands identified in the study have been shown in the literature to express verified experimental antiviral activity, and this study sheds light on their plausible mechanisms of action. While the in-silico methodology employed in this study is comprehensive and supports reported evidence of potential antiviral activity for some hit ligands, future studies are required to validate activity and clinical efficacy. The study focuses on a specific set of compounds and interactions, which may limit its generalizability to other drug candidates or viral strains. Discussing the broader implications of the findings and potential applicability to related viruses or drug targets would enrich the manuscript. Thank you for this important observation. While drug repurposing is a well-known drug discovery strategy that gained high attention and applicability during the past few decades, this research represents a rational drug discovery project that focuses on a specific protein-protein interaction for the challenging new virus. COVID-19, in an attempt to overcome potential future outbreaks and devastating pandemic. The generalizability of this approach is highlighted; specifically, the benefits of utilizing in silico platforms towards a repurposing strategy have been highlighted in the text. It is important to note that our hit compounds were selected based on a detailed analysis of their pharmacological profiles and suitability for translation towards a clinical setting. While a number of similar studies report compounds based mainly on favorable binding scores we carefully filtered through the hit compounds and determined those with complementary pharmacological and safety profiles. It is this comprehensive approach we hope will improve the clinical translatability of our findings. It would be beneficial for the authors to provide information on the availability of the datasets, software codes, and molecular structures used in the study. Enhancing data accessibility would promote transparency and reproducibility in computational drug discovery research. Thank you for this comment, we have now included the following section to the materials and methods: The crystal structures analysed during the current study are available in the PDB database, [PDB ID: 6M0J]”. The molecular structures of the compound’s datasets used were obtained from DrugBank database (https://go.drugbank.com/drugs) and Zinc database (https://zinc.docking.org/substances/subsets/fda/) While the selected compounds show promise in silico, their clinical feasibility and safety profiles need to be thoroughly evaluated. Discussion of potential side effects, pharmacokinetic properties, and drug interactions would provide a more comprehensive assessment of their suitability for clinical trials. We thank the reviewer for this helpful comment. We have accordingly included additional data on the pharmacokinetic profiles for the top hit ligands. It is important to note that the current research utilized ligands from worldwide approved and USFDA approved drugs and nutraceuticals, all of which are clinically used medications or natural compounds with known drug-drug interactions, pharmacokinetic, and toxicity profiles. We have in the discussion highlighted the clinical translatability and significance of repurposing drugs such as Lymecycline and Hexoprenaline in the clinical setting COVID-19 and their plausible applicability. The clinical feasibility and limitations of select hit compounds are discussed in light known clinical and experimental findings. Our repurposing approach in this study aims to gives the work more translatability towards a clinical setting." } ] } ]	1	https://f1000research.com/articles/12-1452
https://f1000research.com/articles/13-840/v1	26 Jul 24	{ "type": "Study Protocol", "title": "Efficacy of Mat Pilates on the resting blood pressure and health related quality of life in individuals with systemic hypertension versus standard care: study protocol for a single centered single blinded randomized controlled trial", "authors": [ "Nivedita S. Prabhu", "G. Arun Maiya", "Vaishali K", "Shivashankara KN", "Nivedita S. Prabhu", "G. Arun Maiya", "Vaishali K" ], "abstract": "Background Systemic hypertension is a global non-communicable disease that creates an essential need for alternate forms of lifestyle modifications, including exercise, to lower elevated blood pressure. Mat Pilates, a feasible form of mind-body coordinated exercise, may provide benefits with limited resources.\n\nObjective This randomized controlled trial aimed to assess the efficacy of Mat Pilates on resting blood pressure and health-related quality of life in individuals with systemic hypertension.\n\nMethods A two-arm, single-blinded, block randomized controlled trial will be recruited with120 participants into control and experimental groups (1:1 ratio). Mat Pilates will be administered to the experimental group and standard care to the control group for 12 weeks. Resting and central blood pressure, health-related quality of life, and echocardiographic parameters will be measured before and after the trial. We hypothesized that Pilates may not be beneficial in lowering resting blood pressure in hypertensive patients. A repeated-measures analysis of variance (ANOVA) will test the within- and between-group effects of Mat Pilates on various outcomes.\n\nEthics and Dissemination The trial was approved by the Institutional Research Committee, Manipal College of Health Professions, Manipal Academy of Higher Education, Kasturba Medical College, and Kasturba Hospital Institutional Ethics Committee, Manipal. Written informed consent will be obtained from all the participants. All stakeholders and committees will communicate key findings regarding the implementation of mind-body association exercises as a measure of lifestyle modification in individuals with systemic hypertension.\n\nTrial registration Clinical Trials Registry of India: CTRI/2021/07/035002. Registered on July 20, 2021, http://ctri.nic.in.", "keywords": [ "exercise", "blood pressure", "quality of life", "Pilates", "movement therapy" ], "content": "Article summary\n\nStrengths and Limitations of this study\n\n• This single-blinded, randomized controlled trial will enable awareness of a feasible, cost-effective form of Mat Pilates exercise for participants diagnosed with hypertension that can be easily implemented in low-resource settings.\n\n• As a mind-body association exercise, individuals with systemic hypertension would benefit from relaxed breathing maneuvers, which will in turn aid in the relaxation of various muscle groups and peripheral vasculature.\n\n• As this is a single-center trial, this study may not be accessible to participants outside the study setting. In addition, Mat Pilates needs supervision for a minimum of 2 weeks to ensure the correct learning of exercise patterns.\n\n\nIntroduction\n\nSystemic arterial hypertension is a significant contributor to the non-communicable disease spectrum. The World Health Organization (WHO) reports that systemic hypertension accounts for almost 45% and 51% of mortality from heart disease and stroke worldwide, respectively.1 The Global Action Plan for the control and prevention of non-communicable diseases (NCDs) aims to reduce the prevalence of systemic hypertension by the year 2025.2 The South Asian subcontinent is undergoing extensive economic growth, leading to inimical urban lifestyle changes. A mortality rate of 1.5 million deaths per year has been reported due to cardiovascular disease (CVD) among Indians, and by 2020, CVD will be the leading cause of mortality and morbidity.3,4 Therapeutic modes adopted for systemic hypertension include antihypertensive drug therapy, lifestyle modifications such as diet, and regular physical activity or exercise. Dietary Approaches to Stop Hypertension (DASH) and regular aerobic exercise for 30 minutes per day are mandated determinants to lower elevated blood pressure.5–7 Meta-analyses of various randomized controlled trials report dual benefits of aerobic and resistance exercise in lowering blood pressure.8,9 However, these studies sparsely include alternate approaches of mind-body exercises such as Pilates, Tai Chi, Yoga, etc.\n\nThe incorporation of the Pilates method of exercise differs from routine structured physical exercise protocols, as it is based on a mind-body association. A thought process used positively to execute a particular movement with specific breathing patterns, generating kinesthetic awareness aids in reducing mental stress, anxiety, enhancing sleep, and a sense of well-being. Changes in body movement as a whole with concentration, synchronized breathing, and precise control of the body part to be moved with precision in a rhythmic manner creates an environment of the movement pattern and ability to identify the muscle being recruited to perform the desired movement.10–14 Pilates can be performed either on the mat or using Pilates equipment using one’s body weight.15,16 It has been effectively reviewed on various health-related parameters such as body composition,17 stress urinary incontinence,18 and breast cancer survivors’ health-related quality of life,19 falls, and physical fitness in the elderly.20–22\n\nFollowing the principles of control, breathing, concentration, flow of movement, centering, and Pilates have often been used to improve physical function and muscle conditioning. The benefits of Mat Pilates being a feasible, novel, and recreational mode of exercise implementing cost-effective resources of mats has been reported in different populations such as multiple sclerosis, Parkinson’s disease, older adults, and low back pain.23–26 Energy expenditure (EE), blood lactate levels, oxygen uptake (VO2 uptake), and low cardiovascular stress (including heart rate and blood pressure) have been observed with the implementation of Mat Pilates when compared to the Reformer Pilates method (which utilizes larger equipment) in healthy young female adults.27 Although the above studies report beneficial effects of Mat Pilates on a spectrum of disease conditions, there is a scarcity of studies reporting the effects of mat Pilates on variations in peripheral and central blood pressure parameters in individuals with systemic hypertension. In addition, structural and hemodynamic changes within the cardiac chambers have rarely been reported in relation to this exercise method in hypertensive individuals.\n\nThe primary and secondary objectives of this trial were to study the effects of mat Pilates on.\n\nPrimary outcomes:\n\n1. Resting blood pressure (systolic and diastolic blood pressure) (mm of Hg)\n\n2. Central systolic blood pressure and pulse pressure (mm of Hg)\n\n3. Mean arterial pressure (MAP) (mm of Hg)\n\nSecondary outcomes:\n\n4. Health related quality of life (HrQoL)\n\n5. Two-dimensional (2D) echocardiographic parameters included the left ventricular ejection fraction (LVEF) and end-diastolic volume.\n\nHypotheses:\n\nNull hypothesis: This trial hypothesizes that mat Pilates will not have a beneficial reduction in arterial blood pressure and will not improve health-related quality of life in participants with systemic hypertension.\n\nAlternate hypothesis: Mat Pilates has a positive effect on reducing resting blood pressure, including central pulse pressure and central systolic pressure. In addition, the application of mat Pilates will improve health-related quality of life in individuals with systemic hypertension.\n\nConsidering the fewer studies reported, this trial will be a two-arm, single-blinded, prospective randomized controlled trial (RCT) aimed at evaluating the efficacy of a 12 weeks, mat Pilates program on changes in resting and central blood pressures (central pulse pressure and central systolic pressure) in a total of 120 participants.\n\nParticipants will be screened in the outpatient departments of General Medicine and Cardiology, Kasturba Hospital, Manipal.\n\nMat Pilates exercises will be administered and supervised in the outpatient department of the Physiotherapy and Health Performance Laboratory, Kasturba Hospital Manipal.\n\nParticipants aged between 30 and 60 years diagnosed with systemic arterial hypertension with optimal blood pressure control on regular antihypertensive therapy will be recruited for this clinical trial.\n\nInclusion criteria:\n\n1. Stage 1 (systolic blood pressure > 130–139 mmHg or diastolic blood pressure > 80–89 mmHg) and Stage 2 (systolic blood pressure >140–159 mmHg or diastolic blood pressure > 90–99 mmHg) hypertensive individuals.\n\nExclusion criteria:\n\n1. Stage 2 hypertension: systolic blood pressure >160 mmHg and diastolic blood pressure >100 mmHg.\n\n2. Accelerated hypertension/Hypertensive crisis.\n\n3. Any discontinuation or change in the antihypertensive drug regime during the study period.\n\n4. Diagnosed cases of acute myocardial infarction, valvular heart disease (aortic, mitral, tricuspid, pulmonic), arterial aneurysms with impending cardiac arrhythmias (on external or internal pacing devices, implanted cardioverter defibrillator (ICD).\n\n5. Pregnancy and lactating hypertensive women.\n\n6. Individuals with cancer on previous or current chemotherapy, radiation therapy.\n\n7. Acute or chronic renal insufficiency on peritoneal or hemodialysis.\n\n8. Individuals with neurological disease causing impaired cognition and incoordination of movements.\n\n9. Musculoskeletal trauma with disability restricting movement patterns.\n\n10. Individuals with vertigo\n\n11. Individuals unable to comprehend instructions for the exercise program.\n\n\nMethods\n\nThe study protocol was approved by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee, Kasturba Hospital, Manipal (an associate hospital of the Manipal Academy of Higher Education, Manipal, Karnataka, India) (registration number: ECR/146/Inst/KA/2013/RR-19) to recruit participants (IEC: 596/2019. Date of approval September 11, 2019). The trial has been registered under the Clinical Trials Registry of India (CTRI/2021/07/035002. Registered on July 20, 2021, http://ctri.nic.in.) This clinical trial adheres to the guidelines and ethical principles for medical research of the Declaration of Helsinki involving human participants.\n\nAny protocol-related amendments during the trial tenure will be communicated to the Institutional Research Committee, Institutional Ethics Committee, Directorate of Research, Manipal Academy of Higher Education, and Clinical Trial Registry of India. Written informed consent will be obtained from all the participants. Stakeholders and committees will receive key findings regarding the implementation of mind-body association exercises as a measure of lifestyle modification in individuals with systemic hypertension. Post-trial ancillary care will be provided to participants in the control group after completion of the clinical trial period if they wish to undergo intervention group exercises.\n\nThe calculated sample size was based on the primary outcome of resting BP considering a level of significance (α) of 0.05, with a power of 80%. With an anticipated standardized difference of four standard deviations of the primary outcome, the estimated minimal clinically important difference (MCID) at 2 mmHg after a period of 12 weeks (at baseline and after 12th week) in both the control and experimental groups, a sample of 44 participants in each group was calculated. Considering an attrition of 20%, the sample size was calculated to be 55 participants and rounded up to 60 participants per group owing to block randomization.\n\nA random number sequence will be generated using computerized random number generator. Participants will be randomized to the control and intervention groups with 1:1 allocation and a block size of 6 consisting of ten participants in each block per group. The allocation sequence will be generated using computer-generated randomized allocation software. Using the sequentially numbered opaque envelope (SNOSE) method, allocation concealment is performed. Of the 120 participants, 60 will be allocated to the control or experimental group.\n\nThis RCT will be a single-blind study. Random allocation to both groups will be performed by a health professional who is not a member of the investigator team. At the completion of twelve weeks, a blinded outcome assessor will assess set outcomes of the respective groups.\n\nPrior permission was sought from the copyright publishers for the administration of the World Health Organization (WHO) BREF Health-Related Quality of Life Questionnaire in two languages. Participants will be screened for eligibility based on their diagnosis and stage of systemic hypertension. Eligible participants will then be familiarized with the study using the participant information sheet, and willing participants will be recruited after obtaining their written informed consent. Preliminary screening will be implemented and written informed consent will be obtained at the outpatient clinic of the Department of Medicine, Kasturba Hospital, Manipal, Karnataka, India.\n\nBaseline assessment of three resting blood pressure measurements in supine lying position with a rest period of one minute in between measurements will be recorded at the same time of the day (10 am). Participants will be instructed to abstain from consumption of alcohol, caffeine, or any caffeine-containing beverage 10 hours prior to the exercise testing session. The values of central systolic blood pressure, central pulse pressure, and mean arterial pressure will be measured by the principal investigator. Left ventricular ejection fraction (LVEF) and left ventricular end-diastolic volume will be measured by a professional expert trained in echocardiography. Including demographic data, the baseline scores of the WHO BREF HRQOL, anthropometric measurements, and exercise stress testing will be measured. This time point will be denoted as T0. Baseline assessment of eligible participants will be performed at the Health Performance Laboratory, Department of Physiotherapy, Clinical Teaching Center, and echocardiographic measurements will be recorded at the Department of Cardiology, Kasturba Hospital, Manipal, India. The participants will then be sequentially block randomized into two groups by a blinded investigator.\n\nParticipants in the experimental group will undergo a two-week supervised Mat Pilates program, after which exercises will be performed at home for 10 weeks. An exercise instruction manual for the mat Pilates program will be provided to every participant at the end of 2 weeks. A weekly telephonic, supervised monitoring call will be implemented to address and record any issues related to compliance, adherence, or adverse events. In addition, they will be advised to follow standard care with the recommended salt intake of 2.4 g) or sodium chloride (6 g) per day and regular physical activity of 3-5 days per week.28\n\nThe control group will receive standard care and a prescribed antihypertensive drug regimen. After 12 weeks, the outcomes will be reassessed and recorded (T1) by a blinded investigator. Adherence will be set at 75% of the total number of sessions (27 of 36 sessions). A flowchart of the procedure is shown in Figure 1. The schedule of participant enrolment, interventions, and assessments has been provided (Figure 2) according to the Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) guidelines.29\n\nT0, T1 Time points at baseline (T0) and after 12 weeks (T1); BP, blood pressure; CPET, cardiopulmonary exercise stress test; HrQoL, health related quality of life; LV, left ventricle; LVEF, left ventricular ejection fraction; RPE, rating of perceived exertion; SNOSE, sequentially numbered opaque sealed envelope; WHOQOL-BREF, World Health Organization Quality of Life Brief version.\n\nT0, T1, Time points at baseline (T0) and after 12 weeks (90 days) (T1); 2D Echocardiography, Two dimensional echocardiography; LV, Left ventricular; HRQoL, health related quality of life.\n\nThe Mat Pilates exercise program will consist of 11 different exercises with a warm-up of 15 min and cool down for 10 min. Three sessions per week of 60 minutes each will be implemented (Table 1). The exercises will be familiarized with, taught, and monitored by a certified mat Pilates instructor.\n\nAdverse events will be recorded and reported every week during telephonic monitoring. Participants will be questioned regarding any major or minor events that may have occurred during the course of their exercise program tenure. The reporting of events, if any, will be implemented by the Institutional Ethics committee for further proceedings.\n\nThe measuring equipment used will be standardized and calibrated periodically every 3 months. The Diamond® Aneroid (BPDL-250 Dial Deluxe) pressure monitor with field calibration feature will be used to measure the resting blood pressure. Mats of six feet by four feet with one and a half inch thickness will be used to perform mat Pilates exercises. The Microlife® WatchBP Office Central, which is an oscillometric calibrated blood pressure device, will measure central blood pressure parameters non-invasively using an arm cuff. Additionally, this device could detect any ongoing atrial fibrillation in the participant. The balance protocol for exercise stress testing will be performed on a Stayfit® testing treadmill.\n\nPrimary\n\nResting blood pressure values (systolic/diastolic) in mmHg will be assessed at T0 and T1. Central blood pressure variables (systolic/diastolic/pulse) will be assessed non-invasively pre- and post-intervention, indicating target organ perfusion and perfusion pressure in the central organs (cerebrum, myocardium, kidneys, and liver). Changes observed in central blood pressure with regular exercise would aid in identifying any progression or reduction in target organ health in systemic hypertension.\n\nSecondary\n\nHealth-related quality of life (HrQOL) in the mental, physical, and psychosocial domains will be assessed at T0 and T1, indicating any variation in the function of daily living in individuals with systemic hypertension. Two-dimensional echocardiographic findings (LVEF, left ventricular end diastolic volume) as secondary outcomes will aid in identifying any structural, ventricular wall stress-related changes in the myocardial wall before and after 12 weeks of the mat Pilates program versus standard care.\n\nData will be statistically analyzed using the Jamovi software. Descriptive analysis will be used to interpret demographics, intention-to-treat analysis will be used for missing data, and subgroup analysis based on age will be used to interpret age-wise changes in blood pressure. The Kolmogorov–Smirnov test will be used as a test of normality, and repeated-measures analysis of variance (ANOVA) will be used to analyze the within-group and between-group differences and pre- and post-differences at T0 and T1. Analysis of covariance (ANCOVA) will be used to analyze the contribution of covariates in the study. All analyses of recorded data will comply with the standards of data analysis for randomized controlled trials that include the analysis of missing data due to attrition.\n\n\nDiscussion\n\nThis randomized controlled trial study protocol highlights the effect of a 12 weeks mat Pilates exercise program on various blood pressure variables. This is one of the first studies to report the efficacy of Pilates exercises and changes in central blood pressures. The strengths of this study include changes in central and peripheral blood pressures, which may reduce cardiovascular stress induced in individuals with systemic hypertension. Changes in left ventricular volumes and myocardial wall thickness could be determined, thereby aiding in a cost-effective method to exercise in hypertensive individuals. Increased central blood pressure causes target organ hypoperfusion, leading to early end organ failure.\n\nAs this trial is a prospective, single-blinded study, the probability of selection bias would be reduced. An added advantage of a home-based program after a two week supervised mat Pilates exercise program would support participants in comprehending the need to exercise better, making this program cost-effective, feasible, and reduce frequent center-based visits. Although home-based, lack of supervision during the exercise regime may be a limitation of this trial. Secondary limitations would include communication between participants in both groups, which could alter exercise behavior, and alternate options for training at other exercise centers. However, if this RCT proves to be effective, it will be the one of the first studies to assess the efficacy of Mat Pilates on central blood pressure variables and ventricular remodeling related to probable changes in ventricular mass-to-volume ratios.\n\n\nEthics approval and consent to participate\n\nThe study protocol was approved by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee, Kasturba Hospital, Manipal (an associate hospital of the Manipal Academy of Higher Education, Manipal, Karnataka, India) (registration number: ECR/146/Inst/KA/2013/RR-19) to recruit participants (IEC: 596/2019. Date of approval September 11, 2019). The trial has been registered under the Clinical Trials Registry of India (CTRI/2021/07/035002. Registered on July 20, 2021, http://ctri.nic.in.) This clinical trial adheres to the guidelines and ethical principles for medical research of the Declaration of Helsinki involving human participants.\n\nAny protocol-related amendments during the trial tenure will be communicated to the Institutional Research Committee, Institutional Ethics Committee, Directorate of Research, Manipal Academy of Higher Education, and Clinical Trial Registry of India. Written informed consent will be obtained from all the participants. Stakeholders and committees will receive key findings regarding the implementation of mind-body association exercises as a measure of lifestyle modification in individuals with systemic hypertension. Post-trial ancillary care will be provided to participants in the control group after completion of the clinical trial period if they wish to undergo intervention group exercises.\n\n\nConsent for publication\n\nNot applicable.\n\n\nPatient or public involvement\n\nPatients or the public were not involved in designing, conducting, or disseminating methods in our research.\n\n\nAuthor contributions\n\nNSP conceptualized the study concept, objectives, design, performed a preliminary and ongoing literature review, and drafted the manuscript. SKN is a clinical expert in the field of systemic hypertension who performed a literature review and scrutinized the final draft of the manuscript. GAM and VK aided in the study conception, scrutinizing, and preliminary drafting of the manuscript.\n\n\nAuthors’ information\n\nNSP is currently an Assistant Professor pursuing her PhD in the Department of Physiotherapy, Manipal College of Health Professions, Manipal Academy of Higher Education with more than 12 years of teaching and research experience in the field of cardiac and pulmonary sciences. SKN is currently Professor in the Department of Medicine, Kasturba Medical College, Manipal Academy of Higher Education and has over 25 years of research and clinical expertise in the area of non-communicable diseases such as hypertension and diabetes mellitus. AGM is currently Professor and Dean, Department of Physiotherapy, Manipal College of Health Professions, Manipal Academy of Higher Education with more than 30 years of research experience in the field of diabetes mellitus and rehabilitation. VK is currently Professor, Department of Physiotherapy, Manipal College of Health Professions, Manipal Academy of Higher Education, with over 25 years of experience in the areas of geriatrics and pulmonary rehabilitation.", "appendix": "Data availability statement\n\nNo data were associated with this article.\n\nThe World Health Organization Quality of Life BREF Questionnaire (English Version) 30 will be used for this study for participants fluent in English and Kannada version will be used for participants fluent in Kannada, a regional language from the state of Karnataka. 31\n\nDataverse: SPIRIT Guidelines checklist: Checklist for Efficacy of Mat Pilates on the resting blood pressure and health related quality of life in individuals with systemic hypertension versus standard care: study protocol for a single centered single blinded randomized controlled trial, DOI: https://doi.org/10.7910/DVN/GLMROL.\n\n\nAcknowledgements\n\nThe authors acknowledge the research and technical expertise provided by the clinical experts of the departments of Cardiology and Cardiovascular Technology, Kasturba Hospital, Manipal, Karnataka, India for 2D echocardiography measurements and interpretation, and the Department of Data Sciences, Prasanna School of Public Health, Manipal Academy of Higher Education for providing statistical analysis, sample size, and attrition rate calculation. The authors acknowledge the valuable contribution and guidance regarding the statistical analysis provided by Dr. Ravishankar N from the Department of Biostatistics, Vallabhbhai Patel Chest Institute, Delhi University, Delhi.\n\n\nReferences\n\nCauses of Death 2008 [online database]. Geneva: World Health Organization. Reference SourceReference Source\n\nGlobal action plan for the prevention and control of noncommunicable diseases 2013-2020. http\n\nGaziano T, Reddy KS, Paccaud F, et al.: Cardiovascular disease.Jamison DT, Mosley WH, editors. Disease Control Priorities in the Developing World. Oxford: Oxford University Press; 2006; p. 645–62.\n\nWorld Health Organization: The World Health Report 2002: Reducing Risk, Promoting Healthy Life. Geneva, Switzerland: World Health Organization; 2002.\n\nSacks FM, Svetkey LP, Vollmer WM, et al.: Effects on blood pressure of reduced dietary sodium and the Dietary Approaches to Stop Hypertension (DASH) diet. DASH-Sodium Collaborative Research Group. N. Engl. J. Med. 2001; 344: 3–10. Publisher Full Text\n\nKelley GA, Kelley KS: Progressive resistance exercise and resting blood pressure: A meta-analysis of randomized controlled trials. Hypertension. 2000; 35: 838–843. Publisher Full Text\n\nWhelton SP, Chin A, Xin X, et al.: Effect of aerobic exercise on blood pressure: A meta-analysis of randomized, controlled trials. Ann. Intern. Med. 2002; 136: 493–503. Publisher Full Text\n\nReddy KS, Prabhakaran D, Chaturvedi V, et al.: Methods for establishing a surveillance system for cardiovascular diseases in Indian industrial populations. Bull. World Health Organ. 2006; 84: 461–469. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHypertension Study Group: Prevalence, awareness, treatment and control of hypertension among the elderly in Bangladesh and India: A multicenter study. Bull. World Health Organ. 2001; 79: 490–500.\n\nLange C, Unnithan VB, Larkam E, et al.: Maximizing the benefits of Pilates-inspired exercise for learning functional motor skills. J. Bodyw. Mov. Ther. 2000; 4: 99–108. Publisher Full Text\n\nPilates J, Miller W: Return to life through contrology. Incline Village: Presentation Dynamics.1945.\n\nPilates JH: Your health. Incline Village: Presentation Dynamics.1934.\n\nLatey P: The Pilates method: history and philosophy. J. Bodyw. Mov. Ther. 2001; 5: 275–282. Publisher Full Text\n\nLessen D, The PMA: Pilates certification exam study guide. Miami: Pilates Method Alliance; 2014.\n\nAnderson B: Fitting Pilates into a rehabilitation practice. Rehab Manag. 2010; 23(24): 26–27.\n\nKloubec J: Pilates: how does it work and who needs it? Muscles Ligaments Tendons J. 2011; 1: 61–66. PubMed Abstract\n\nAladro-Gonzalvo AR, Machado-Díaz M, Moncada-Jiménez J, et al.: The effect of Pilates exercises on body composition: a systematic review. J. Bodyw. Mov. Ther. 2012; 16: 109–114. PubMed Abstract \| Publisher Full Text\n\nBo K, Herbert RD: There is not yet strong evidence that exercise regimens other than pelvic floor muscle training can reduce stress urinary incontinence in women: a systematic review. J. Physiother. 2013; 59: 159–168. PubMed Abstract \| Publisher Full Text\n\nStan DL, Collins NM, Olsen MM, et al.: The evolution of mindfulness-based physical interventions in breast cancer survivors. Evid Based Complementary Altern Med. 2012; 2012: 1–15. Publisher Full Text\n\nGranacher U, Gollhofer A, Hortobagyi T, et al.: The importance of trunk muscle strength for balance, functional performance, and fall prevention in seniors: a systematic review. Sports Med. 2013; 43: 627–641. PubMed Abstract \| Publisher Full Text\n\nCancela JM, de Oliveira IM , Rodriguez FG: Effects of Pilates in physical fitness on older adults. A systematic review. Eur. Rev. Aging Phys. Act. 2014; 11: 81–94. Publisher Full Text\n\nBarker AL, Bird ML, Talevski J: Effect of Pilates exercise for improving balance in older adults: a systematic review with meta-analysis. Arch. Phys. Med. Rehabil. 2015; 96: 715–723. PubMed Abstract \| Publisher Full Text\n\nBulguroglu I, Guclu-Gunduz A, Yazici G, et al.: The effects of Mat Pilates and Reformer Pilates in patients with Multiple Sclerosis: A randomized controlled study. NeuroRehabilitation. 2017 Jan 1; 41(2): 413–422. PubMed Abstract \| Publisher Full Text\n\nSuárez-Iglesias D, Miller KJ, Seijo-Martínez M, et al.: Benefits of Pilates in Parkinson’s disease: a systematic review and meta-analysis. Medicina. 2019 Aug; 55(8): 476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBertoli J, Biduski GM, de la Rocha FC : Six weeks of Mat Pilates training are enough to improve functional capacity in elderly women. J. Bodyw. Mov. Ther. 2017 Oct 1; 21(4): 1003–1008. PubMed Abstract \| Publisher Full Text\n\nDa Luz MA Jr, Costa LO, Fuhro FF, et al.: Effectiveness of mat Pilates or equipment-based Pilates exercises in patients with chronic nonspecific low back pain: a randomized controlled trial. Phys. Ther. 2014 May 1; 94(5): 623–631. PubMed Abstract \| Publisher Full Text\n\nde Souza AL , da Silva AI , Mochizuki L, et al.: What is the exercise intensity of Pilates? An analysis of the energy expenditure, blood lactate, and intensity of apparatus and mat Pilates sessions. J. Bodyw. Mov. Ther. 2021 Apr 1; 26: 36–42. Publisher Full Text\n\nHypertension Study Group: Prevalence, awareness, treatment and control of hypertension among the elderly in Bangladesh and India: A multicenter study. Bull. World Health Organ. 2001; 79: 490–500.\n\nChan A-W, Tetzlaff JM, Gøtzsche PC, et al.: SPIRIT 2013 Explanation and Elaboration: Guidance for protocols of clinical trials. BMJ. 2013; 346: e7586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan A-W, Tetzlaff JM, Altman DG, et al.: SPIRIT 2013 Statement: Defining Standard Protocol Items for Clinical Trials. Ann. Intern. Med. 2013; 158: 200–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrabhu N: SPIRIT Guidelines checklist. Harvard Dataverse. 2024; V1. Publisher Full Text\n\nWHOQOL: Measuring Quality of Life, “WHOQOL-BREF”. http" }	[ { "id": "308430", "date": "21 Aug 2024", "name": "Shakeel Ahmed", "expertise": [ "Reviewer Expertise Pulmonary rehabilitation", "Respiratory neuroplasticity", "Cardiovascular Rehabilitation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 objective sentence is incomplete. Insert \"is\" after trial in the sentence.\nMethods: rephrase to \"A two-arm, single-blinded, block randomized controlled trial will recruit 120 participants\"\nArticle summary: Strength and Limitations: 1. 1st and 2nd points contradict your hypothesis stated in the abstract.\nProcedure: Elaborate on what constitutes standard care for control group? What about physical activity? can the participants in the control group exercise at home or a gym ? is this controlled?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "317117", "date": "09 Sep 2024", "name": "Susana Lopes", "expertise": [ "Reviewer Expertise Pilates", "hypertension", "cardiac rehabilitation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study protocol The aims to investigate the effectiveness of Mat Pilates in reducing blood pressure and improving quality of life in individuals with hypertension. A randomized controlled trial will compare the effects of Mat Pilates to standard care over 12 weeks. The primary outcome measures include resting and central blood pressure, health-related quality of life, and echocardiographic parameters. This is a well written and easy to read manuscript. it is interesting, however, some issues could be addressed.\nSpecific comments\nAbstract: Objective . Where we read \"this RCT aimed to\", should read \"this RCT aims to \" A two-arm, single-blinded, block randomized controlled trial will be recruited with120 participants into control and experimental groups (1:1 ratio). - Is confusing. Rephrase\nIntroduction: Epidemiological data would benefit with more up to date references. Authors state a scarcity of studies studying the effects of pilates on blood pressure, but many studies have been published recently including systematic reviews and meta-analysis .\nObjectives - secondary outcomes Why did the authors choose \"Two-dimensional (2D) echocardiographic parameters included the left ventricular ejection fraction (LVEF) and end-diastolic volume\" as a secondary outcome for this population? Do you expect to see structural changes in the heart and ejection fraction in a 12-week mat pilates program?\nEligibility criteria Why is systolic blood pressure >160 a exclusion criteria, since systolic blood pressure < 180 mm hg are usually eligible for exercise?\nMust there not be an interval before starting exercise while medication needs to be implemented. I mean, medication should be unchanged at least 1 month before starting exercise to avoid any change in blood pressure values due to medication\nwhy was a 2mmHg difference used to estimate sample size?\nI have some questions regarding the interventions. Will the exercise group not be taking any anti-hypertensive medication? Why is it just referred for the control group? This must be clarified.\nHow to you intend to control for anti-hypertension medication intake? and to other exercise regimes in both groups?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/13-840
https://f1000research.com/articles/13-432/v1	01 May 24	{ "type": "Research Article", "title": "Knowledge, attitudes, and practices of Lebanese patients with type II diabetes towards the use and abuse of dietary supplements: A cross-sectional study", "authors": [ "Maher Abdallah", "Sahar Dandachy", "Nour Ahmad", "Marwa Sleiman", "Rania Mansour", "Maha Hoteit", "Maher Abdallah", "Sahar Dandachy", "Nour Ahmad", "Marwa Sleiman" ], "abstract": "Background Dietary supplements (DS) use among Lebanese patients with type 2 diabetes mellitus (T2DM) increased widely due to the country’s economic and financial situation. This study was conducted (1) to estimate the prevalence of DS use among persons with T2DM amid the escalating economic crisis in Lebanon; (2) to explore the knowledge, attitude, and practice (KAP) of DS use; and (3) to determine any significant association between socio-economic and socio-demographic factors and the use of DS modality\n\nMethods A cross-sectional study was conducted during the worst episode of the economic crisis between October and April 2022 on 460 adult patients with T2DM of both sexes. Patients were interviewed using a pre-tested questionnaire.\n\nResults Almost 4 out of 10 patients with T2DM in our study were found to be using DS, where 27.6% take multivitamins frequently. One-third of the participants agreed that nutritional supplements are necessary to control diabetes symptoms and complications. Around 41.1% of the participants complained about hypoglycemia and used DS to control their blood sugar levels (56.4%), while the rest used it to improve their health (35.5%) and control their diet (2.2%). The predictors of DS usage were the patient’s level of education [OR=3.9, CI=1.5-10, p=0.003), self-monitoring of blood sugars (OR=4.9, CI=1.68-14.6; p=0.004) and reading the nutrition label [OR=59.3, CI=6.3-55.8, p=0.000].\n\nConclusion This study estimated the prevalence of DS use and abuse, among persons with diabetes type II and found three significant predictors of DS use among patients with T2DM. Public health experts should encourage healthy discussions and awareness with their patients to comprehend their views regarding DS use.", "keywords": [ "Lebanon", "type 2 diabetes", "KAP", "dietary supplements" ], "content": "1. Introduction\n\nAmong the sustainable developmental goals (SDG), SDG 3.4 addresses Non-Communicable Diseases (NCDs) specifically, and world leaders committed to a one-third reduction in deaths between the ages 30 to 70 years from diabetes by the year 2030. Although the burden is worldwide, low- and middle-income countries (LMICs) are struggling with NCDs, with over three-quarters of all NCD deaths occurring in these countries.1 For instance, in Lebanon, 91% of all deaths are attributed to NCDs,2 and the initiatives, led by the Ministry of Public Health, to address the NCD burden failed to be implemented adequately.3–5 This is due to the country’s political and economic challenges.6 On the other hand, Lebanese patients with type 2 diabetes mellitus (T2DM) are experiencing medicine shortages,7 which leads the patients to be incapable of buying their medical drugs. This deviation increased the health risk for these patients and increased the use and abuse of dietary supplements (DS). This increase may be due to several factors, including patients’ perceptions that DS’s natural products are safer, more effective, and cheaper than conventional medicines.8 In Lebanon, there are no studies that investigate the prevalence of DS use and abuse among persons with T2DM or their knowledge, attitudes, and practices toward DS use. Our research group had already investigated DS use among children under 5 and their mothers,9 among adults during the COVID-19 pandemic,10 and among athletes.11 Thus, the aims of the current study are: (1) to estimate the prevalence of DS use among persons with T2DM amid the escalating economic crisis in Lebanon; (2) to explore the knowledge, attitude, and practice (KAP) of DS use; and (3) to determine any significant association between socio-economic and socio-demographic factors and the use of DS modality.\n\n\n2. Methods\n\nA cross-sectional study was conducted between August 1 and November 2022 across the 4 main governorates in Lebanon (Beirut, Mount Lebanon, Beqaa, and North Lebanon). This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. After the approval of the ethical committee at al Zahraa University Medical Center (#157/May 7, 2022), we approached the medical files of patients with T2DM to retrieve their phone numbers from many hospitals, outpatient departments, and private clinics in Lebanon. Patients signed a written consent form before being enrolled in the study.\n\nConvenient sample size was collected from all governorates in Lebanon. A total of 460 patients with T2DM were enrolled in this survey. The response rate was 100%.\n\n2.2.1 Inclusion and exclusion criteria\n\nThe criteria for inclusion of patients in the current study were: participants with T2DM who are older than 18 years and have given their informed consent to take part in the study. The exclusion criteria were individuals under the age of 18 years because of the high probability of having type 1 diabetes mellitus at this age. In addition, pregnant women with gestational diabetes, elderly people, and people with intellectual disabilities were excluded from this study.\n\nUsing a standardized questionnaire, a face-to-face interview between licensed dietitians and the patients was conducted. To ensure the validity of the data collection tool, the questionnaire was adapted from relevant literature in English, then translated by experts into Arabic and back-translated into English to check the translation.12 The data was gathered using the administered questionnaire in Arabic. The questionnaire was made to find out the participants’ KAP on DS use as well as to identify the prevalence and most popular DS modalities in Lebanon. It was divided into five sections: (1) demographics, (2) clinical data about T2DM, (3) knowledge, (4) attitude, and (5) practice of DS therapies. All information about the patient’s demographics, including gender, age, marital status, level of education, and employment status, was recorded. Additionally, the time of the first time being diagnosed with T2DM, the type of T2DM therapy (insulin or oral medicines), the complications related to T2DM, and other co-morbid issues are clinical factors that were included in the study. In the knowledge section, we checked the knowledge of patients with T2DM with regard to DS efficacy and safety concerns. Moreover, in the attitude part, participants were asked about their attitudes toward the use of DS. Would they, for example, abide by their doctor’s recommendations to avoid using DS. Would they consult their physicians before using DS or not. In the practice section, we asked patients with T2DM if they had ever used DS for diabetes specifically. Participants who replied “yes” were then questioned about the type of DS used, who prescribed it, whether they were informed by their physician about it, whether they combined it with their T2DM medications, and whether they had ever used DS for a condition other than T2DM.\n\nThe data was coded and checked for completeness and consistency. All responses from the questionnaire were entered into Microsoft Excel, and a quality check was performed; data cleaning. Then it was exported to SPSS. Statistical analysis was conducted using SPSS (IBM Corp, SPSS Statistics version 26) (https://www.ibm.com/support/pages/spss-statistics-260-fix-pack-1). SPSS was used for data entry, coding, data management, and analysis. The results were described as frequencies and percentages for variables. The associations between both the demographic factors and the clinical data about T2DM with DS were determined using Pearson’s Chi-square test. A p value of ≤ 0.05 was considered significant. Odds ratio was considered as a measure of strength. Multivariate logistic regression was used to identify predictors of DS usage.\n\n\n3. Results\n\nFour hundred and sixty subjects participated in this study. The frequency distribution of their socio-demographic findings is presented in Table 1. Most of the participants were aged 41-60 years (51.3 %), males (51.3 %), from North Lebanon (49.1%), married (60.2%), had a university degree (39.8%), unemployed (41.3%), and had no monthly income (35.4%)\n\nThe clinical data characteristics of the study participants with T2DM are shown in Table 2. Most of the patients conducted their laboratory tests three months prior to the data collection (37%), and around 34% were newly diagnosed with T2DM. Furthermore, approximately 32% of the study participants developed diabetic complications such as neuropathy (5.7%), nephropathy (5.7%), diabetic foot syndrome (7.2%), and retinal disease (13.5%), which was the most common. Most of the study participants had a family history of T2DM (88.7%), most of them inherited T2DM from their fathers (31.3%). As for medical treatment, the vast majority of patients (72%) were on oral anti-diabetic medications, and most of them had other medical comorbidities such as coronary artery disease (21.3%), osteoporosis (9.3%), kidney disease (8.7%), and hypertension (23.7%).\n\na Retinal eye disease, nasal allergy, ear disease, liver disease, foot disease, polycystic ovary syndrome, thyroid, asthma, gastrointestinal disorders, and thrombocytopenia.\n\n3.3.1 Knowledge\n\nFigure 1 shows the participants’ knowledge towards the use of DS among persons with T2DM during the economic crisis. It appears that most of them (85.9%) had heard about DS, more than half of the sample (63%) knew that DS had efficacity, and 67% believed that DS are safe as shown in Figure 1.\n\nDS, Dietary supplements.\n\n3.3.2 Attitudes\n\nThe participants’ attitudes regarding DS use are presented in Figure 2. It was shown that 81% of patients would first discuss DS use with their physician and that the majority (85.7%) would not use it if their physicians didn’t recommend it. On the other hand, more than half of participants (57.6%) suggested the same DS to a family member as shown in Figure 2. Table 3 displays the results of the participants’ attitudes based on a Likert scale scoring system consisting of responses of strongly agree, agree, neutral, disagree, and strongly disagree. “Strongly agree” and “agree” responses were combined to show the total percentage of “good attitude,” “neutral” for “not aware”, while “disagree” and “strongly disagree” were also combined to show the total percentage for “poor attitude.” One-third of the participants agreed that nutritional supplements can control the management of diabetes and prevent further complications such as retinal disease, foot disease, kidney disease, and nerve damage. On the contrary, only 34% of patients disagreed that nutritional supplements are a suitable substitute for a healthy and balanced diet in the treatment of diabetes. Furthermore, more than 44% disagreed that nutritional supplements are as effective as medication in modifying blood sugar levels in diabetic patients.\n\nDS, Dietary supplements.\n\n3.3.3 Practices\n\nAmong persons with T2DM, the estimated prevalence of DS use during the escalating crisis was 41.1% as shown in Figure 3. It appears that more than half the participants used previously DS to prevent other medical complications and combine DS with their diabetes medications. This use was always controlled by their physicians rather than using it on their own as shown in Figure 4. Multivitamins (27.6%) and vitamin C (22.4%) were the most DS used daily (Table 4). Furthermore, per monthly and/or weekly use, vitamin D (11.3%), ginger supplements (14.3%) and green tea supplements (7.4%) were the most recorded DS (Table 4). According to Table 5, around 41.1% of the participants complain about hypoglycemia and used DS to control their blood sugar levels (56.4%), while the rest used it to improve their health (35.5%) and control their diet (2.2%). What’s more, it has been demonstrated that 65% of the participants always read the DS label before ingesting it and 67.6% will use always continue using the DS (Table 5).\n\nDS, Dietary supplements; T2DM, type 2 diabetes mellitus.\n\nDS, Dietary supplements; T2DM, type 2 diabetes mellitus.\n\nBased on the bivariate analysis, we attempted to determine the extent of the contribution of the variables of interest to the probability of dietary supplements use among patients with T2DM using the logistic regression analysis. Table 6 shows that the gender, age, residency, marital status, monthly income, being employed, being on diet, having other comorbidities, length of diabetes disease, family history, knowledge about the efficacity and safety of DS and the source of information regarding DS were not considered as predictors for the use of DS among T2MD patients. On the other hand, the level of education can mediate the use of DS. For instance, those who are university graduates ranked first among DS consumers compared to those who are uneducated or above [OR=3.9, CI=1.5-10, p=0.003). In addition, patients with T2DM who monitor less their blood sugars have 5 times higher odd to buy more DS compared to their counterparts (OR=4.3, CI=1.78-10.8; p=0.001). Furthermore, patients who read never the label were more prone to buy DS compared to patients who always read the label [OR=28.2, CI=3.5-225, p=0.002].\n\n\n4. Discussion\n\nTo the best of our knowledge, this is the first study that investigates the prevalence, and correlates of DS use among patients with T2DM during the economic crisis and drug shortage time in Lebanon. Almost 4 out of 10 patients with T2DM in our study were found to be using and abusing DS. Compared to the Unites states of America (USA), it was shown that 6 out of 10 Americans with T2DM use DS.13 The prevalence reported in our study was higher than that reported in Saudi Arabia (17.6%),14 Australia (24%),15 and Thailand (5.3%)16 but lower than the prevalence observed in Canada (44%),17 and in 3 studies from the United States of America (USA) (62.1%; 67%; 58%).18–20\n\nThe most common DS used daily in the current study were multivitamins (27.6%), vitamin C (22.4%), followed by vitamin D (21.5%), iron (18%), and calcium (17.4%). These findings came hand in hand with the data reported in a Canadian study17 (27.5% multivitamins, 18.9% vitamin E, 18.7% vitamin C, and 16% calcium), but it contradicts the results reported in Clifford et al., where in Australia vitamin C (18%) ranked first followed by garlic (17%), omega 3 (14%), vitamin E (13%) and multivitamins (12%).15\n\nAccording to the literature, the use of DS for diabetes management has always been reviewed from a pharmacy standpoint and from that of complementary and alternative medicine.21 However, as supplement use continues to grow in Lebanon,9–11 it is important for healthcare professionals to understand the evidence behind prescribing supplements and their potential role as part of medical care especially during the unstable conditions. For instance, according to the literature review, meta-analyses assessing the impact of vitamin C supplementation on diabetes-related outcomes assumed an improvement of fasting blood glucose only without any improvement in HbA1c in patients with T2DM.22–24 In addition, three meta-analyses that examined folate or folic acid supplementation had conflicting findings.25–27 As for B12 supplementation, many studies showed that individuals taking Metformin suffer from depleted serum B12 levels, and human studies on both B6 and biotin were extremely limited, with a lack of narrative reviews on both vitamins’ impact on T2DM patients.28 On the other hand, a meta-analysis that assessed the niacin supplementation showed an increased risk of T2DM onset following supplementation.29 As for vitamin D, a review conducted by Li et al. showed that most studies in patients with T2DM used vitamin D at 2000 IU/day, which may improve glycemic control and a dose of 4000 IU/day may be elicited to provide positive effects on HbA1c, HOMA-IR, and the fasting plasma glucose.30\n\nThe DS used by our study population was prescribed mainly by their physicians (70.2%). However, in the Arabian Gulf states, patients with T2DM did not reveal, discuss, or even seek medical advice from a physician, which differed from our findings, where participants relied heavily on physicians for DS advice. For instance, out of six studies conducted in the Gulf Club Countries (GCC), the majority of DS users did not tell their physicians about the use of DS.14,31–35 Similarly, in Nigeria and the USA where the majority of DS among patients with T2DM were not being taken based on a recommendation from a health provider.13,36\n\nAlmost more than half of the participants in our study were satisfied with their DS use and intended to use it again (67.6%). Unlike Saudi Arabian participants with T2DM, where only 6.7% of respondents said they would use DS again, and 55.7% regretted its use.14 In our study, 85% of the participants had heard of DS, some of them (67.4%) believed DS were safe, and more than half (63%) knew that DS are effective where 33.9% agreed that DS are necessary to control diabetes and 31% strongly agreed that DS prevent diabetic complications. These findings align with the data reported in an updated literature review that showed that patients usually expressed the attitude that DS may not help much but will not hurt.37 Additionally, our participants’ main reason for utilizing DS in this study was to lower their blood sugar level (56.1%), followed by improving overall health (35.5%). Thus, a responsible healthcare approach is much needed for the patients to receive evidence-based DS information about efficacy, effectiveness, adverse effects, and possible interactions. A slight majority (57.2%) of participants supported the use of DS in conjunction with their medical treatment for T2DM. This finding aligns with the result observed in a Saudi Arabian and Nigerian study where 90% and 67% of the patients with T2DM preferred combining DS with their conventional therapies, respectively.14,36\n\nIn a qualitative study in Pakistan, the principal motivator of DS use was the desire to cure T2DM, where 41% preferred combining DS and T2DM.38\n\nIn our study, the level of education, the frequency of monitoring blood sugars, and reading labels can mediate the use of DS. On contrary, the predictors of DS use in Saudi Arabia were age above 51 years, unemployment, and the participants’ knowledge about the effectiveness of complementary and alternative medicine (CAM) products.14 Moreover, in Thailand, female gender, age 40-69 years, and diabetes duration of less than 10 years were significant correlators of DS use.39 A Chinese study found that DS use among people with T2DM was associated with a history of previous DS use for other conditions, a positive attitude towards DS, efficacy of DS, and a longer duration of diabetes.40\n\nMoreover, a study from Malaysia found that females were 1.8 times more likely to use DS than males.41 Furthermore, a study from Bahrain showed that females DS users were more likely to be dominating, and those who have had diabetes for a longer time and have T2DM complications were the top users.42 Another study from Saudi Arabia showed that the most common users of DS practices were older females, housewives, and illiterates.43\n\nThis study presents some limitations. First, it lacks the impact of DS on T2DM compared to conventional therapy. Second, a self-reported questionnaire was used for most of the reported measures. Thus, bias may be present due to inaccurate self-reporting and memory in some questions. Third, this study was of cross-sectional survey; therefore, only associations can be determined and not causations. Notably, the strength of this study is that it is the first study that has been carried out in Lebanon which brought up the topic of knowledge, attitudes, and practices to DS use among patients with T2DM.\n\n\n5. Conclusion\n\nThis study estimated the prevalence of DS use and abuse among patients with T2DM during the time of medicine shortage and economic crisis. Public health experts should encourage healthy discussions with their patients to comprehend their views regarding DS use. In addition, clinicians and researchers should collaborate to initiate safety and efficacy trials on common DS used for diabetes. Accordingly, relevant institutions whether governmental and non-governmental organizations, are strongly asked to design awareness programs that will be addressed to target groups and implemented by a specialized team in which social workers and health promoters play an important role by developing materials which fit all the perception of all categories that were shown by the study, and especially suitable to those who are illiterate, and who cannot read labels. The presence of evidence-based studies in the form of randomized controlled trials will help both patients and clinicians regarding the use of a DS product.\n\n\nEthics and consent\n\nThe study was approved by the ethical committee of al Zahraa University Medical Center (#157/May 7, 2022). Written informed consent was obtained from all subjects involved in the study. Written informed consent was obtained also from the patient(s) to publish this paper.\n\n\nAuthors’ contributions\n\nMA, RM and MH conceived the idea, designed the study, helped in collecting the data, analyzed the data, drafted and reviewed the manuscript. SD helped in collecting the data, reviewed and edited the manuscript. NA and MS collected and analyzed the data. All authors read and approved the final manuscript.", "appendix": "Data availability\n\nOpen Science Framework: Dietary Supplement and Diabetes type II. https://doi.org/10.17605/OSF.IO/ZVNJD. 44\n\nThis project contains the following underlying data\n\n- Dietary supplement and diabetes spss.sav\n\n- STROBE checklist\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nGlobal Week for Action on NCDs: (accessed on 13 April 2023). Reference Source\n\nWHO EMRO: Health of Refugees and Migrants 2018. (accessed on 13 April 2023). Reference Source\n\nNon-Communicable Diseases Prevention and Control Plan (NCD-PCP) Lebanon 2016-2020. Accessed 13 Apr 2023. Reference Source\n\nWorld health Organization: PRIMARY HEALTH CARE SYSTEMS (PRIMASYS) Comprehensive case study from Lebanon 2017. Google Search. Accessed 13 Apr 2023. Reference Source\n\nDoocy S, Lyles E, Hanquart B, et al.: Prevalence, care-seeking, and health service utilization for non-communicable diseases among Syrian refugees and host communities in Lebanon. Confl. Heal. 2016; 10: 1–17.\n\nZablith N, Diaconu K, Naja F, et al.: Dynamics of non-communicable disease prevention, diagnosis and control in Lebanon, a fragile setting. Confl. Heal. 2021; 15: 1–13.\n\nDas M: Lebanon faces critical shortage of drugs. Lancet Oncol. 2021; 22: 1063. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNyenwe EA, Jerkins TW, Umpierrez GE, et al.: Management of type 2 diabetes: evolving strategies for the treatment of patients with type 2 diabetes. Metabolism. 2011; 60: 1–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohsen H, Ibrahim C, Bookari K, et al.: Prevalence of Essential Nutrient Supplement Use and Assessment of the Knowledge and Attitudes of Lebanese Mothers towards Dietary Supplement Practices in Maternal, Infancy and Preschool Ages: Findings of a National Representative Cross-Sectional Study. Foods. 2022; 11: 3005. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohsen H, Yazbeck N, Al-Jawaldeh A, et al.: Knowledge, attitudes, and practices related to dietary supplementation, before and during the COVID-19 pandemic: Findings from a cross-sectional survey in the Lebanese population. Int. J. Environ. Res. Public Health. 2021; 18: 8856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadek Z, Mohsen H, Yazbek S, et al.: Dietary Supplements Use among Athletes in Lebanon: Knowledge, Attitudes, Practices, and Correlates. Foods. 2022; 11: 1521. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRovira M-A, Grau M, Castañer O, et al.: Dietary supplement use and health-related behaviors in a Mediterranean population. J. Nutr. Educ. Behav. 2013; 45: 386–391. PubMed Abstract \| Publisher Full Text\n\nWilson PB: Population-representative analysis of dietary supplementation among Americans with diabetes mellitus. J. Diabetes. 2019; 11: 115–121. PubMed Abstract \| Publisher Full Text\n\nAl-Eidi S, Tayel S, Al-Slail F, et al.: Knowledge, attitude and practice of patients with type 2 diabetes mellitus towards complementary and alternative medicine. J. Integr. Med. 2016; 14: 187–196. PubMed Abstract \| Publisher Full Text\n\nClifford RM, Batty KT, Davis W, et al.: Prevalence and predictors of complementary medicine usage in diabetes: Fremantle diabetes study. J. Pharm. Pract. Res. 2003; 33: 260–264. Publisher Full Text\n\nSripa S: Usage of and cost of complementary/alternative medicine in diabetic patients. J. Med. Assoc. Thail. 2005; 88: 1630–1637.\n\nRyan EA, Pick ME, Marceau C: Use of alternative medicines in diabetes mellitus. Diabet. Med. 2001; 18: 242–245. Publisher Full Text\n\nArcury TA, Bell RA, Snively BM, et al.: Complementary and alternative medicine use as health self-management: rural older adults with diabetes. J. Gerontol. Ser. B Psychol. Sci. Soc. Sci. 2006; 61: S62–S70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarrow D, Egede LE: Association between complementary and alternative medicine use, preventive care practices, and use of conventional medical services among adults with diabetes. Diabetes Care. 2006; 29: 15–19. PubMed Abstract \| Publisher Full Text\n\nLi J, Li X, Gathirua-Mwangi W, et al.: Prevalence and trends in dietary supplement use among US adults with diabetes: the National Health and Nutrition Examination Surveys, 1999–2014. BMJ Open Diabetes Research and Care. 2020; 8: e000925. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYilmaz Z, Piracha F, Anderson L, et al.: Supplements for diabetes mellitus: a review of the literature. J. Pharm. Pract. 2017; 30: 631–638. Publisher Full Text\n\nAshor AW, Werner AD, Lara J, et al.: Effects of vitamin C supplementation on glycaemic control: a systematic review and meta-analysis of randomised controlled trials. Eur. J. Clin. Nutr. 2017; 71: 1371–1380. PubMed Abstract \| Publisher Full Text\n\nde Paula TP , Kramer CK, Viana LV, et al.: Effects of individual micronutrients on blood pressure in patients with type 2 diabetes: a systematic review and meta-analysis of randomized clinical trials. Sci. Rep. 2017; 7: 40751. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhodaeian M, Tabatabaei-Malazy O, Qorbani M, et al.: Effect of vitamins C and E on insulin resistance in diabetes: a meta-analysis study. Eur. J. Clin. Investig. 2015; 45: 1161–1174. Publisher Full Text\n\nAkbari M, Tabrizi R, Lankarani KB, et al.: The effects of folate supplementation on diabetes biomarkers among patients with metabolic diseases: a systematic review and meta-analysis of randomized controlled trials. Horm. Metab. Res. 2018; 50: 93–105. PubMed Abstract \| Publisher Full Text\n\nSudchada P, Saokaew S, Sridetch S, et al.: Effect of folic acid supplementation on plasma total homocysteine levels and glycemic control in patients with type 2 diabetes: a systematic review and meta-analysis. Diabetes Res. Clin. Pract. 2012; 98: 151–158. PubMed Abstract \| Publisher Full Text\n\nZhao JV, Schooling CM, Zhao JX: The effects of folate supplementation on glucose metabolism and risk of type 2 diabetes: a systematic review and meta-analysis of randomized controlled trials. Ann. Epidemiol. 2018; 28: 249–257.e1. PubMed Abstract \| Publisher Full Text\n\nYan MK-W, Khalil H: Vitamin supplements in type 2 diabetes mellitus management: A review. Diabetes Metab. Syndr. 2017; 11: S589–S595. Publisher Full Text\n\nVerdoia M, Schaffer A, Suryapranata H, et al.: Effects of HDL-modifiers on cardiovascular outcomes: a meta-analysis of randomized trials. Nutr. Metab. Cardiovasc. Dis. 2015; 25: 9–23. PubMed Abstract \| Publisher Full Text\n\nLi X, Liu Y, Zheng Y, et al.: The effect of vitamin D supplementation on glycemic control in type 2 diabetes patients: a systematic review and meta-analysis. Nutrients. 2018; 10: 375. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Rowais NA: Herbal medicine in the treatment of diabetes mellitus. Saudi Med. J. 2002; 23: 1327–1331. PubMed Abstract\n\nAl Saeedi M, El Zubier AG, Bahnassi AA, et al.: Patterns of belief and use of traditional remedies by diabetic patients in Mecca, Saudi Arabia. East Mediterr. Health J. 2003; 9(1-2): 99–107. PubMed Abstract \| Publisher Full Text\n\nAl-Garni AM, Al-Raddadi RM, Al-Amri TA: Patterns and determinants of complementary and alternative medicine use among type 2 diabetic patients in a diabetic center in Saudi Arabia: herbal alternative use in type 2 diabetes. J. Fundam. Appl .Sci. 2017; 9: 1738–1748.\n\nKamel FO, Magadmi RM, Hagras MM, et al.: Knowledge, attitude, and beliefs toward traditional herbal medicine use among diabetics in Jeddah Saudi Arabia. Complement. Ther. Clin. Pract. 2017; 29: 207–212. PubMed Abstract \| Publisher Full Text\n\nBakhotmah BA, Alzahrani HA: Self-reported use of complementary and alternative medicine (CAM) products in topical treatment of diabetic foot disorders by diabetic patients in Jeddah, Western Saudi Arabia. BMC. Res. Notes. 2010; 3: 1–8.\n\nAmaeze OU, Aderemi-Williams RI, Ayo-Vaughan MA, et al.: Herbal medicine use among type 2 diabetes mellitus patients in Nigeria: understanding the magnitude and predictors of use. Int. J. Clin. Pharm. 2018; 40: 580–588. PubMed Abstract \| Publisher Full Text\n\nChang H, Wallis M, Tiralongo E: Use of complementary and alternative medicine among people living with diabetes: literature review. J. Adv. Nurs. 2007; 58: 307–319. PubMed Abstract \| Publisher Full Text\n\nBukhsh A, Gan SH, Goh B-H, et al.: Complementary and alternative medicine practices among type 2 diabetes patients in Pakistan: a qualitative insight. Eur. J. Integr. Med. 2018; 23: 43–49. Publisher Full Text\n\nWanchai A, Phrompayak D: Use of complementary and alternative medicine among Thai patients with type 2 diabetes mellitus. J. Integr. Med. 2016; 14: 297–305. PubMed Abstract \| Publisher Full Text\n\nChang H-YA, Wallis M, Tiralongo E: Predictors of complementary and alternative medicine use by people with type 2 diabetes. J. Adv. Nurs. 2012; 68: 1256–1266. PubMed Abstract \| Publisher Full Text\n\nChing SM, Zakaria ZA, Paimin F, et al.: Complementary alternative medicine use among patients with type 2 diabetes mellitus in the primary care setting: a cross-sectional study in Malaysia. BMC Complement. Altern. Med. 2013; 13: 1–7.\n\nKhalaf AJ, Whitford DL: The use of complementary and alternative medicine by patients with diabetes mellitus in Bahrain: a cross-sectional study. BMC Complement. Altern. Med. 2010; 10: 1–5.\n\nElolemy AT, AlBedah AM: Public knowledge, attitude and practice of complementary and alternative medicine in Riyadh region, Saudi Arabia. Oman Med. J. 2012; 27: 20–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoteit M: Dietary Supplement and Diabetes type II. [Dataset]. OSF. 2024. Publisher Full Text" }	[ { "id": "275466", "date": "10 May 2024", "name": "Rabih Hallit", "expertise": [ "Reviewer Expertise Infectious disease and internal medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this paper. Dear authors, Thank you for this paper. I have some comments that you might want to take into account in order to improve its quality: -The introduction is too short and can be easily expanded. You did not introduce the idea of dietary supplements, the knowledge, attitude and practice about these supplements according to previous international studies. -Rationale is missing: why is it important to conduct this study in patients with diabetes? The fact that the same study was conducted among patients with other diseases is not sufficient. -Your first objective needs to be introduced. What is the relation of the economic crisis in Lebanon with your study and with the use of dietary supplements in patients with diabetes? -In section 2.3, how did you build the questionnaire? based on previous studies or you created your own scale? I suggest adding the questionnaire as an appendix with the right answers for the knowledge questions most importantly. -The results lack the bivariate analyses. Please add them and then you enter in the multivariable model all variables that showed a p < 0.25 in the bivariate analysis only. -The discussion is good but it needs adjustment maybe after implementing the changes to the results. -Please add the clinical implications paragraph to the discussion: how would other healthcare professionals benefit from your study?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "11560", "date": "26 Jun 2024", "name": "Maha Hoteit", "role": "Author Response", "response": "Thank you for inviting me to review this paper. Dear authors, Thank you for this paper. I have some comments that you might want to take into account in order to improve its quality: -The introduction is too short and can be easily expanded. You did not introduce the idea of dietary supplements, the knowledge, attitude and practice about these supplements according to previous international studies. Author Response: Done -Rationale is missing: why is it important to conduct this study in patients with diabetes? The fact that the same study was conducted among patients with other diseases is not sufficient. Author Response: Done -Your first objective needs to be introduced. What is the relation of the economic crisis in Lebanon with your study and with the use of dietary supplements in patients with diabetes? Author Response: Done -In section 2.3, how did you build the questionnaire? based on previous studies or you created your own scale? I suggest adding the questionnaire as an appendix with the right answers for the knowledge questions most importantly. Author Response: It was built based on previous published studies with no major changes and all the tools are already available on: Hoteit M: Dietary Supplement and Diabetes type II. [Dataset]. OSF. 2024. Publisher Full Text (reference 44 in the manuscript). As for the responses to knowledge, attitudes and practices, they are already mentioned in the Figures and tables in the manuscript itself. -The results lack the bivariate analyses. Please add them and then you enter in the multivariable model all variables that showed a p < 0.25 in the bivariate analysis only. Author Response: Bivariate analyses are already shown in Table 6. -The discussion is good but it needs adjustment maybe after implementing the changes to the results. Author Response: Done -Please add the clinical implications paragraph to the discussion: how would other healthcare professionals benefit from your study? Author Response: Done" } ] }, { "id": "275467", "date": "21 Jun 2024", "name": "Abeer Salman Alzaben", "expertise": [ "Reviewer Expertise Nutrition" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for providing me the opportunity to review the manuscript. below some of my comments to make the manuscript more clear:\nIntroduction :\n·\n\nIntroduction is too short and lack of the rational. If there is shortage in medicine in Lebanon. why the authors worry about DS?\n\n. may be need some information about % Dm in Lebanon or DS use ? ·\n\nDoes the objective to assess DS among all individuals with DM or only Type 2?\n\nMethods :\n·\n\nPlease correct “convenient sample size was collected from all governorates in Lebanon. A total of 460 patients with T2DM were enrolled in this survey to “convenient sample technique” Inclusion/ exclusion criteria, ·\n\n“The exclusion criteria were individuals under the age of 18 years because of the high probability of having type 1 diabetes mellitus at this age.” Some individuals maybe older than 18 with type 1. Are those excluded? ·\n\nWhat about other disease such as Celiac or food allergies. Are those excluded? ·\n\nRegarding the KSP. More information is needed about scoring. does the questions with one option ·\n\nRegarding the use of DS, I feel the questions/information are not clear. Do you mean they are currently use? Past use? If past use, how frequent? If a person has taken 1 pill in the last year. How you consider this? Need more clarifications.\nResults :\n\n·\n\nTable 1: You had 14 % illiterate. Do you think those are significantly different in KSP and DS use than others? ·\n\nDuration of Disease newly diagnosis vs < 5 years. I would say 1- 5 years are not newly diagnosis. Can you separate them as one subgroup? ·\n\nFor me, it is still not clear, all sample with type 2 DM? Did you exclude type 1? ·\n\nFigure 1, x axis refers to % but the sum does not = 100%, have you heard about DS is = 86 %? Figure 1 there are 2 option yes and don’t know what about no ? In addition, the knowledge items that have been asked are not knowledge questions, maybe awareness? ·\n\nBe consistent with decimals\n\n·\n\nFigure 2 not clear. what does the bar stand for? I think you need to work on all your figure legends. explain what X and Y axis means, it refers to n number or %. ·\n\nHow many participants have never used DS? ·\n\nTable 6 in education, I did not see the illiterate group in the Determinants.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11943", "date": "26 Jul 2024", "name": "Maha Hoteit", "role": "Author Response", "response": "Introduction : - Introduction is too short and lack of the rational. If there is shortage in medicine in Lebanon. why the authors worry about DS? Thank you for this comment. The introduction was amended based on your comments. We are worried about DS use because Lebanese patients with type 2 diabetes mellitus (T2DM) are experiencing medicine shortages with rise in medication prices, which leads the patients to be incapable of buying their medical drugs. This deviation increased the health risk for these patients and increased the use and abuse of dietary supplements (DS). This increase may be due to several factors, including patients' perceptions that DS's natural products are safer, more effective, and cheaper than conventional medicines. This was mentioned in lines 56-60. -May be need some information about % Dm in Lebanon or DS use ? There is no information concerning DS use among patients with diabetes type 2 in Lebanon but we already mentioned that we already assessed DS use among: children under 5 and their mothers, adults and athletes. This was added in lines 62-64. - Does the objective to assess DS among all individuals with DM or only Type 2? This is mentioned in rationale, objectives and methodology: patients with type 2 diabetes only. -Methods : - Please correct “convenient sample size was collected from all governorates in Lebanon. A total of 460 patients with T2DM were enrolled in this survey to “convenient sample technique” The whole paragraph was amended. Inclusion/ exclusion criteria, “The exclusion criteria were individuals under the age of 18 years because of the high probability of having type 1 diabetes mellitus at this age.” Some individuals maybe older than 18 with type 1. Are those excluded? Yes these patients were also excluded. This was amended in the same paragraph. What about other diseases such as Celiac or food allergies. Are those excluded? This was not mentioned as an exclusion criterion. However, none of our patients were having these diseases because we were collecting the medical history of the patients. All patients who were excluded were having abnormal levels of glycemia, confirmed having type 2 diabetes and not diabetes type 1. Regarding the KSP. More information is needed about scoring. does the questions with one option. As mentioned in the “Methodology” section, the questions were having multiple responses for instance the Table 3 displays the results of the participants’ attitudes based on a Likert scale scoring system consisting of responses of strongly agree, agree, neutral, disagree, and strongly disagree. There was no single score, but we were showing the response of the patients. We are planning in future studies to validate this score. Regarding the use of DS, I feel the questions/information are not clear. Do you mean they are currently use? Past use? If past use, how frequent? If a person has taken 1 pill in the last year. How you consider this? Need more clarifications. We already mention in “Methodology” that we are assessing the current use of the DS by the patients. The answers of these questions are mentioned in Table 4 showing the frequency of the current use by the patients (per day, week, month…). The use of pills was most considered compared to the quantity. Based on that the frequency was our main objective. Results : Table 1: You had 14 % illiterate. Do you think those are significantly different in KSP and DS use than others? This was presented in Table 6 which shows that people who are educated had an 1.4-3.9 higher odd of using DS compared to those who are illiterate. · Duration of Disease newly diagnosis vs < 5 years. I would say 1- 5 years are not newly diagnosis. Can you separate them as one subgroup? Newly diagnosed are those who were diagnosed in the same year. The other sub-groups are defined as 1-5 years. This was amended in the manuscript and the tables. · For me, it is still not clear, all sample with type 2 DM? Did you exclude type 1? All patients with DT1 were excluded. And yes only patients with type 2 DM were included in the study. · Figure 1, x axis refers to % but the sum does not = 100%, have you heard about DS is = 86 %? 86% of participants know and heard about DS and 14% don’t know about it. We only highlighted the answer “Yes” and “Don’t Know”. However, we amended Figure 1. Figure 1 there are 2 option yes and don’t know what about no ? In addition, the knowledge items that have been asked are not knowledge questions, maybe awareness? Based on the study source of the questionnaire, these questions were related to knowledge questions. · Be consistent with decimals This was amended in the manuscript. · Figure 2 not clear. what does the bar stand for? I think you need to work on all your figure legends. explain what X and Y axis means, it refers to n number or %. X axis refers to the percentage of patients responding to the questions raised in Y axis. Only “Yes” responses are shown in this Figure. This sentence was added to the Figures in the manuscript to make it more clearer. · How many participants have never used DS? 59% of the population in the current study. This is shown in Figure 3. · Table 6 in education, I did not see the illiterate group in the Determinants. It is available but said “ uneducated”. We will change it to be consistent." } ] } ]	1	https://f1000research.com/articles/13-432
https://f1000research.com/articles/13-525/v1	22 May 24	{ "type": "Research Article", "title": "Clinical and pathological characteristics of blastoid mantle cell lymphoma: a single institution experience", "authors": [ "Vidya Monappa", "Swathi Prabhu", "Ranjini Kudva", "Vishwapriya Mahadev Godkhindi", "Kanthilatha Pai", "Ananth Pai", "Sharada Mailankody", "Vidya Monappa", "Ranjini Kudva", "Vishwapriya Mahadev Godkhindi", "Kanthilatha Pai", "Ananth Pai", "Sharada Mailankody" ], "abstract": "Background Blastoid mantle cell lymphoma (B-MCL) is a rare aggressive lymphoma. It is characterized by blastoid morphology with high proliferation and inconsistent immunohistochemistry (IHC), making it a diagnostic challenge for the pathologist.\n\nMethods This is a retrospective analytical cohort study. We reviewed biopsy confirmed cases of B-MCL diagnosed over a period of 10 years (January 2012 to December 2022). The clinical presentation, histopathological and IHC findings, treatment received, and survival outcomes were studied. Randomly selected cases of classic MCL (n=12), diagnosed during the same period served as controls.\n\nResults A total of 12 cases were studied. Four cases were transformed from previously diagnosed MCL; 8 cases arose de novo. Mean age was 61.17 years and the male: female ratio was 5:1. Half of the cases showed extra nodal extension and 81.8% had bone marrow involvement. Gastrointestinal tract was the most common site of extra nodal involvement. Histopathological examination showed diffuse involvement of the lymph node with medium sized cells. On immunohistochemistry, one of the cases showed loss of CD5 expression while the other had aberrant CD10 expression. Mean Ki-67 index was 58.09% in the cases and 16.33% in controls and was statistically significant (p=0.005). The median overall survival (OS) for cases was 2 years vs 8 years in controls. The p53 over expression (>30% nuclear positivity) was seen in 66.6% cases (4/6).\n\nConclusion There are several factors that contribute to the aggressiveness of B-MCL, and new treatment approaches might be required to improve patient outcomes.", "keywords": [ "Mantle cell lymphoma", "blastoid", "cyclin D1", "lymphoma" ], "content": "Introduction\n\nMantle cell lymphoma (MCL) is a known subtype of B-cell non-Hodgkin lymphoma (NHL), with an aggressive clinical course and outcome. Its incidence is higher in Asians than in western population.1,2 It was first described in the year 1973 when Karl Lennert et al. used the term centrocytic lymphoma due to its resemblance to centrocytes. In 1992, Banks et al. coined the term MCL, which was entrenched in the WHO blue book in 2001, officially recognizing this entity.3 It was named so because of its distinctive growth pattern, called “mantle zone pattern,” wherein, tumor cells encircle and overcrowd the reactive germinal center, replacing it.4 It commonly affects elderly male patients in their 7th decade. Nodal involvement is the most common presentation. However, extra nodal involvement, especially of the gastrointestinal tract, bone marrow, and spleen, is not unusual. MCL molecular pathogenesis is characterized by the signature translocation t (11;14) (q13; q32), involving immunoglobulin heavy chain (IGH) and CCND1 genes (encoding cyclin D1 and regulating the CDK4/6 complex with pro-tumorigenic effects). Apart from this characteristic translocation, SOX11 overexpression, p53 mutations, and lymph node microenvironment contribute to the proliferation and growth of mantle cells attributing to its malignant behavior and aggressive nature.5\n\nMCL accounts for 3 to 10% of NHL of which 1/3rd is blastoid subtype (B-MCL), making it one of the rarest lymphoma subtypes.6,7 B-MCL could arise de novo or transform from a classic subtype, although the former is more common. It is classified by its distinct morphological and immunohistochemical features, which contribute to its aggressive behavior, associated with significant mortality and morbidity.2 The pattern of lymph node involvement in B-MCL varies, predominantly diffuse or predominantly nodular, with a few cases exhibiting a mantle zone-like pattern. Neoplastic cells resemble lymphoblasts with high mitotic activity (≥20–30/10 high-power fields).6 There is no consensus on the treatment of B-MCL. They are usually detected as stage III/IV disease and are managed aggressively owing to the high incidence of relapse rates. The treatment regimen is similar to MCL, with a few modifications depending on the patient status, stage at presentation, and affordability factors.\n\nIn this study, we reviewed 12 cases of biopsy-proven B-MCL and discussed their clinical presentation, morphological and immunohistochemical findings, treatment received, and survival outcomes. Our aim was to identify factors (clinical and pathological) that differentiate B-MCL from classic MCL. This experience will help clinicians devise better treatment strategies, resulting in improved patient survival rates.\n\n\nMethods\n\nThis was a retrospective analytical cohort study. All biopsy-confirmed cases of B-MCL at our institution over a 10-year period (January 2012 to December 2022) were included. The clinical presentation, histopathological and immunohistochemical findings, treatment approaches, and survival outcomes of the patients were studied. Data were extracted from archival and electronic medical records (EMR). A total of 12 B-MCL cases were included in this study. Randomly selected cases of classic MCL (n=12) diagnosed within the same time frame were used as controls. Clinicopathological parameters analyzed included age, sex, nodal and extra nodal involvement, bone marrow involvement (stage IV), and hepatosplenomegaly. B symptoms were included if they were documented previously. The date of diagnosis, treatment received, alive, dead, or lost to follow-up, and any event (relapse/progression) were recorded to facilitate survival studies.\n\nLymphoma panels for CD3, CD5, CD20, CD10, BCL6, BCL2, Ki-67, cyclin D1 were performed in the majority of cases and CD23, TdT, and p53 in selected cases. Loss of IHC markers or aberrant expression, if any, was documented to identify deviations from the expected expression pattern. Ki-67 was assessed by counting the percentage of nuclear positivity in 10 consecutive high-power fields (HPF) in hotspot areas. CD10 and BCL6 were reported to be positive if ≥30% of the cells showed nuclear positivity. BCL2 with ≥50% nuclear expression in the tumor cells was taken as positive. The p53 IHC with nuclear expression >30% was considered as p53 over expression and <30% as low expression, as per the guidelines of the Nordic lymphoma study group.8\n\nThe collected data were analysed using IBM SPSS Statistics for Windows, Version 29.0 (Armonk, NY: IBM Corp). Descriptive statistics, frequency analysis, and percentage analysis were used for categorical variables, and the mean and standard deviation (S.D.) were used for continuous variables. To find a significant difference between the bivariate samples in the independent groups, an independent sample t-test was used. To predict the survival rate, the Kaplan-Meier curve was used with the log-rank method. To find the significance in qualitative categorical data, the chi-square test was used similarly; if the expected cell frequency was less than 5 in 2×2 tables, Fisher’s exact test was used. In all the above statistical tools, a probability value of 0.05 is considered as significant level.\n\n\nResults\n\nThere were 12 cases of biopsy and IHC confirmed B-MCL included in this study. Their ages ranged from 43 to 80 years, with a mean Age at diagnosis of 61.17 years. There were 10 males and 2 females with a male: female ratio of 5:1. Among the 12 cases, four cases (33.3%) were found to have progressed from previous classic MCL, while the remaining 8 cases arose de novo. There were no statistical differences in age or sex when compared with the controls.\n\nAll the patients exhibited nodal involvement. B-MCL cases showed higher rates of extra nodal involvement (50%, n=6/12) when compared to classic MCL (8.3%, n=1/12). Extra nodal sites included the gastrointestinal tract (n=3, 50%), nasopharynx (n=2, 33.3%), and central nervous system (CNS) (n=1, 16.6%). Bone marrow involvement was noted in 9/11 cases and 9/12 controls, respectively. B symptoms were observed in 6/10 cases (60%) while hepatomegaly (n=6/12, 50%), splenomegaly (n=5/10, 50%) were observed more frequently in cases than controls. However, these differences were not statistically significant. The demographic and clinical characteristics of the patients and controls are shown in Table 1.\n\nEffacement of nodal architecture was observed in all cases. Predominantly diffuse pattern (<50% nodular) was observed in 91.6 % of B-MCL(n=11/12), with one case showing predominantly nodular growth pattern (n=1/12,8.4%). Similar growth pattern was observed in control cases as well with slightly lower percentages of predominantly diffuse pattern (n=8/12, 66.6%) and nodular (n=4/12, 33.3%). The p value was not statistically significant (p=0.131), and none of our cases had a mantle zone pattern.\n\nSmall lymphoid cells with slightly irregular nuclear contours and scant cytoplasm were observed in all the classic MCL cases. B-MCL cases showed medium-sized cells with oval nuclei, stippled chromatin, inconspicuous nucleoli, and a scant cytoplasm. This morphology is similar to that of diffuse large B-cell lymphoma (DLBCL) or lymphoblastic lymphoma. Scattered hyalinized blood vessels and histiocytes were observed in both cases and controls. B-MCL demonstrated a high mitotic rate in all cases, exhibiting brisk mitosis (>20/10HPF). In contrast, the control group showed only occasional mitotic figures, and this difference was statistically significant (p<0.0001). Focal necrosis was seen in 2/12 cases (16.6%) and 1/12 controls (8.3%) and was not statistically significant (p=0.537). The histomorphological features of B-MCLs and classic MCL are shown in Table 2.\n\n\n\n• Diffuse (>50%)\n\n\n\n• Nodular (>50%)\n\n\n\n• Small\n\n\n\n• Medium\n\nAll cases were CD20 positive and CD3 negative. CD5 expression was lost in one case (n=1, 8.3%) and aberrant CD10 expression was noted in another case (n=1, 8.3%). All cases and controls showed overexpression of cyclin D1. Furthermore, BCL2 was positive in 83.3% (n=5/6) cases and 100% of controls (n=6/6). BCL6 expression was negative in all cases and controls. CD23 and TdT were performed in a few patients (3 cases, 1 control;4 cases, 2 controls respectively) and were negative.\n\nThe Ki-67 proliferation index was significantly higher (range, 40 to 90%; mean of 58.1%) in the cases compared to the controls (range, 2% to 33%; mean of 16.3%) (Figure 2). This finding was statistically significant, with p value of 0.005. This corresponded to the brisk mitosis noted on histopathology of B-MCL. p53 IHC was available for six cases. Over expression (>30%) nuclear positivity was seen in 4/6 cases (66.6%), while the other two cases showed <30% nuclear positivity (Figure 3). Table 3 highlights the IHC findings in our cases (B-MCL) and the controls (classic MCL).\n\nAn anthracycline-based chemotherapy regimen, along with rituximab comprising cyclophosphamide, adriamycin, vincristine, and prednisolone (R-CHOP), was used as a front-line treatment. Table 4 highlights the treatment and follow-up status of the 12 patients with B-MCLs. The treatment approach was individualized based on patients’ affordability, clinical stage, commutability, and cooperation.\n\n* BR is Bendamustine and rituximab therapy.\n\n** RCHOP stands for Rituximab+cyclophosphamide+dauxorubicin+Vincristine+prednisolone.\n\nThe log-rank for the data in our study was 0.887; thus, the two curves were not statistically significant. This might be due to the small sample size. The survival rates were also influenced by the COVID outbreak which occurred in 2020. The median overall survival for cases was 2 years, and for controls, it was 8 years (Figure 4).\n\n\nDiscussion\n\nMCL usually occurs in the 7th decade with a high male-to-female ratio and advanced clinical stage (stage III/IV). Lymphadenopathy is the most common presentation, but leukemic non-nodal (nnMCL) with bone marrow and splenic involvement has rarely been described. B-MCL, a morphological variant of MCL, is an aggressive subtype with a heterogeneous clinical course and a limited response to standard chemotherapy regimens.6,7\n\nAmong the demographic and clinical characteristics, there were no major differences between B-MCL and classic MCL in our series. Extra nodal involvement was, however, more frequent in B-MCL (n=6, 50%), with GIT being the most common extra nodal site involved. CNS involvement in MCL is associated with elevated Mantle cell lymphoma International Prognostic (MIPI) score, elevated lactate dehydrogenase (LDH) levels, and high Ki-67 index of ≥30%,7 suggesting transformation to a more aggressive subtype during relapse. The One case with CNS involvement in our series showed a high Ki-67 index of 70%, with markedly elevated serum LDH levels. On the contrary only 1/12 of the controls (8.33%) showed extra nodal involvement of GIT. This observation was statistically significant with a p value of 0.024. Extra nodal sites commonly affected in MCL include the gastrointestinal tract (lymphomatous polyposis), CNS, Waldeyer ring, spleen, and bone marrow.6\n\nDuring its course, classic MCL can progress into one of its variants, blastoid (B-MCL) or pleomorphic (P-MCL).1,2,6,9 In this series, 4/12 cases (25%) of B-MCL arose via progression from classic MCL. Khanlari et al. opined that B-MCL and P-MCL are distinct aggressive variants that should be designated separately.2 Hoster et al. observed that Ki-67 index was significantly higher in B-MCLs (80%) vs P-MCLs (39%). Similarly, NOTCH1 mutations are more common in B-MCL than in P-MCL. They also described another hybrid variant of MCL, with blastoid chromatin and a higher degree of pleomorphism. However, because it was more similar to B-MCL, they concluded that it was a variant of B-MCL.10 Blastoid transformation is portended by leukocytosis, high LDH, high proliferation index, histomorphologic changes, diffuse growth pattern, increase in nuclear size, increase in mitosis, and pleomorphism, fulfilling the criteria of B-MCL/P-MCL1,6 (Figure 1). We had four cases which underwent transformation to B-MCL on relapse. One of our cases did show slight pleomorphism, but the Ki-67 was very high (>50%) and was thus called B-MCL.\n\nInset depict the same. Image drawn using iPad pro.\n\nOn immunohistochemistry, MCLs express surface immunoglobulins (IgM, IgD), light chain restriction (lambda), pan B cell markers (CD19, CD20, CD79a), CD5, cyclin D1(>95%). Aberrant loss of CD5 and expression of CD10 and BCL6 are rare but are more common in aggressive variants. We had one case each of CD5 -ve and CD10+ve, while BCL6 was negative in all cases. In their extensive review of CD5 negative MCL, Soleimani et al.11 observed improved survival in these patients independent of other favourable prognostic markers such as SOX11 loss and low Ki-67 and κ light chain restriction. Conversely, this advantage was lost in the presence of a blastoid morphology. Multitude genetic alterations seen in B-MCL (complex karyotype, somatic mutations involving vital genes – TP53, NOTCH1, NOTCH2, CDKN2A, and MUC2 aberrations) were likely to overwhelm good prognostic factors.\n\nXu et al.12 described the clinicopathological and prognostic significance of CD10 expression in 30 MCL cases. They opined that CD10 expression in the more aggressive subsets of MCL – high Ki-67>60%, blastoid/pleomorphic morphology, high MIPI, contributed to worse overall survival (OS) and was statistically significant with p value of <0.05. They also observed a diffuse growth pattern, blastoid/pleomorphic morphology, and BCL6 expression in these cases.12 One of our B-MCL cases showed CD10 expression, with diffuse growth pattern and Ki-67 of 40%, but BCL6 was negative. The literature review showed variable BCL6 positivity in MCL ranging from 11 to 75%.13,14\n\nCyclin D1 negative MCL cases lack cyclin D1 and CCND1 rearrangements and may have CCND2 rearrangements or genetic alterations leading to overexpression of cyclin D2 and cyclin D3 or rarely due to truncated cyclin D1 mRNA.15 SOX11 (>90%) helps to identify CD5 and cyclin D1 negative cases. However, it is not specific for MCL and is also positive in lymphoblastic lymphoma, hairy cell leukemia, and Burkitt lymphoma.14 In this study, we only included cases that were cyclin D1 positive, as SOX11 was not available in our setup. This is probably a limitation of this study.\n\nMCL is generally associated with an aggressive, albeit heterogeneous, clinical course, inadequate response to chemotherapy, and a high recurrence rate with poor long-term prognosis. The blastoid and pleomorphic variants behave even more aggressively, with a median overall survival of 18 months.16 The median OS for cases was 2 years and that for controls was 8 years in this study. Out of 12 cases, 7 cases were dead at the time of this study, with OS ranging from 1 month to 10 years. All four patients who progressed from classic MCL died within a year of B-MCL diagnosis. Mortalities were also observed in the control group (5/12), with four cases clustered around 2020 (peak COVID time in India).\n\nMCL is considered an incurable disease; however, with newer therapeutic approaches, survival has increased to a few years. The prognostic markers for MCL as proposed by WHO for current clinical use incorporates: Age, performance status, CNS involvement at diagnosis, Stage (I, II vs III, IV), S. β2 microglobulin, S. LDH, morphology (Classic vs Blastoid). MCL international prognostic index (MIPI), Ki-67 index (<30% vs >30%), p53 by IHC, TP53 deletions/mutations by sequencing analysis.6 The MIPI is a tailored prognostication index for MCL, based on four independent prognostic factors: age, performance status, LDH, and leukocyte count.10 We could not evaluate MIPI in this study, as none of the necessary factors were documented in the patient files.\n\nThe Ki-67 proliferation index has additional prognostic relevance in MCL.10 A Ki-67 index >30% is associated with worse outcomes.17 Most of our cases had very high Ki-67 index, in the range of 40-90% (Figure 2). Several studies have reported p53 IHC expression in mantle cell lymphomas. The percentage cut off for p53 overexpression as determined by IHC varies across different studies, ranging from 10 to 30 to 50%.5,8,18 In our study, two out of six cases showed low p53 expression. A cut-off of 30% was taken as per Nordic lymphoma study group.8 A p53 IHC expression of more than 50% is associated with poor overall survival of less than 2yrs. A complex karyotype, MYC translocation or overexpression, and unmutated IGHV status are additional poor prognostic markers.5,18\n\nThese are some of the differential diagnoses that we need to consider when dealing with B-MCL; Diffuse large B-cell lymphoma (DLCL) can have cells that resemble blastoid cells of B-MCL. However, they can be ruled out by their negativity for CD5 and cyclin D1. Few cases of DLBCL can express cyclin D1(2%), but they are negative for SOX11.2,19 Lymphoblastic lymphomas must be ruled out because of their close resemblance to the blastoid cells of B-MCL. Nuclear positivity for TdT helps to do the same. It is important to remember that plasma cell myeloma, blastic variant can be cyclin D1 positive (30-40%). However, plasma cells are CD5 and CD20 negative with CD138 positive.20 Occasionally, a starry sky appearance might be observed in Burkitt lymphoma, which is positive for CD10 and BCL6 and negative for CD5 and cyclin D1.6\n\nThere is no established treatment regimen for BMCL. Combined conventional doses of polychemotherapy followed by involved field radiotherapy are available for clinical use. Since MCL patients usually present at an advanced stage, systemic therapy is the standard treatment, with rare surgical intervention in cases presenting with massive splenomegaly or bowel obstruction. However, the response rate with anthracycline based CHOP regimens (cyclophosphamide, doxorubicin, vincristine, and prednisone) was much lower (13-50%) than that of other lymphomas.21 Addition of rituximab (R-CHOP) has been linked to improved survival. Intensive immunochemotherapy with/without allogeneic stem cell transplantation in first remission followed by maintenance and/or consolidation therapies with anti-CD 20 antibodies and/or novel agents including bortezomib, ibrutinib, and lenalidomide have been proposed to improve progression-free and overall survival.5,22\n\n\nConclusion\n\nIn summary, our study demonstrates the poor prognosis of B-MCL patients with higher extra nodal involvement compared to the classic variant. The diagnosis of B-MCL requires meticulous morphological evaluation coupled with comprehensive immunohistochemistry panel. This amalgamation is required for accurately diagnosing B-MCL and to ruling out its morphological mimics. Immunohistochemical variations can be present leading to diagnostic confusion. Furthermore, due to aggressive nature of B-MCL, prompt and tailored therapeutic interventions become imperative. Rarity, aggressiveness, morphological, and immunohistochemical variations with a lack of standard treatment regimens make B-MCL a diagnostic and therapeutic challenge.\n\nThe study did not include cyclin D1 negative cases (non-availability of SOX11). The p value might not be representative because of the low number of cases. Hence, larger study groups are recommended for appropriate values.\n\nThe study has been approved by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC) (Ref: IEC1:325/2023) with date of approval 18/10/2023.\n\n\nConsent\n\nAs the study is retrospective does not involve any intervention of subjects and uses lab based coded data collection; Consent waived by the ethics committee.", "appendix": "Data availability\n\nFigshare: Clinical and pathological characteristics of Blastoid mantle cell lymphoma: a single institution experience. DOI: https://doi.org/10.6084/m9.figshare.25437115.v3. 23\n\nThe project contains the following underlying data:\n\n• BMCL.xlsx. (Anonymised excel sheet of clinical, pathological, immunohistochemical and treatment aspects of cases and controls)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: STROBE-checklist: Clinical and pathological characteristics of Blastoid mantle cell lymphoma: A single institution experience. DOI: https://doi.org/10.6084/m9.figshare.25700322.v5. 24\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nNone.\n\n\nReferences\n\nCortelazzo S, Ponzoni M, Ferreri AJM, et al.: Mantle cell lymphoma. Crit. Rev. Oncol. Hematol. 2020 Sep; 153: 103038. Epub 2020 Jul 4. PubMed Abstract \| Publisher Full Text\n\nKhanlari M, Mo H, Kim DH, et al.Blastoid and Pleomorphic Mantle Cell Lymphomas Demonstrate Distinct Clinicopathologic and Genetic Features. Am. J. Surg. Pathol. 2023 Aug 1; 47(8): 849–858. Epub 2023 Jun 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanks PM, Chan J, Cleary ML, et al.: Mantle cell lymphoma. A proposal for unification of morphologic, immunologic, and molecular data. Am. J. Surg. Pathol. 1992 Jul; 16(7): 637–640. PubMed Abstract \| Publisher Full Text\n\nTiemann M, Schrader C, Klapper W, et al.: Histopathology, cell proliferation indices, and clinical outcome in 304 patients with mantle cell lymphoma (MCL): a clinicopathological study from the European MCL Network. Br. J. Haematol. 2005 Oct; 131(1): 29–38. PubMed Abstract \| Publisher Full Text\n\nJain P, Wang ML: Mantle cell lymphoma in 2022-A comprehensive update on molecular pathogenesis, risk stratification, clinical approach, and current and novel treatments. Am. J. Hematol. 2022 May; 97(5): 638–656. PubMed Abstract \| Publisher Full Text\n\nWHO Classification of Tumours Editorial Board: Hematolymphoid tumors [Internet; beta version ahead of print (in progress)]. Lyon (France) International Agency for Research on Cancer; 2022. [cited Jun 22, 2023]. (WHO Classification of Tumors Series, 5th ed.). Reference Source\n\nShrestha R, Bhatt VR, Guru Murthy GS, et al.: Clinicopathologic features and management of blastoid variant of mantle cell lymphoma. Leuk. Lymphoma. 2015; 56(10): 2759–2767. PubMed Abstract \| Publisher Full Text\n\nNordström L, Sernbo S, Eden P, et al.: SOX11 and TP53 add prognostic information to MIPI in a homogenously treated cohort of mantle cell lymphoma--a Nordic Lymphoma Group study. Br. J. Haematol. 2014 Jul; 166(1): 98–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArgatoff LH, Connors JM, Klasa RJ, et al.: Mantle cell lymphoma: a clinicopathologic study of 80 cases. Blood. 1997 Mar 15; 89(6): 2067–2078. PubMed Abstract \| Publisher Full Text\n\nHoster E, Rosenwald A, Berger F, et al.: Prognostic Value of Ki-67 Index, Cytology, and Growth Pattern in Mantle-Cell Lymphoma: Results From Randomized Trials of the European Mantle Cell Lymphoma Network. J. Clin. Oncol. 2016 Apr 20; 34(12): 1386–1394. PubMed Abstract \| Publisher Full Text\n\nSoleimani A, Navarro A, Liu D, et al.: CD5-negative mantle cell lymphoma: clinicopathologic features of an indolent variant that confers a survival advantage. Leuk. Lymphoma. 2022 Apr; 63(4): 911–917. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu J, Medeiros LJ, Saksena A, et al.: CD10-positive mantle cell lymphoma: clinicopathologic and prognostic study of 30 cases. Oncotarget. 2017 Dec 15; 9(14): 11441–11450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZanetto U, Dong H, Huang Y, et al.: Mantle cell lymphoma with aberrant expression of CD10. Histopathology. 2008; 53: 20–29. PubMed Abstract \| Publisher Full Text\n\nGao J, Peterson L, Nelson B, et al.: Immunophenotypic variations in mantle cell lymphoma. Am. J. Clin. Pathol. 2009 Nov; 132(5): 699–706. PubMed Abstract \| Publisher Full Text\n\nSalaverria I, Royo C, Carvajal-Cuenca A, et al.: CCND2 rearrangements are the most frequent genetic events in cyclin D1(-) mantle cell lymphoma. Blood. 2013 Feb 21; 121(8): 1394–1402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwerdlow SH, Campo E, Pileri SA, et al.: The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 2016 May 19; 127(20): 2375–2390. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCondoluci A, Rossi D, Zucca E, et al.: Toward a Risk-Tailored Therapeutic Policy in Mantle Cell Lymphoma. Curr. Oncol. Rep. 2018 Aug 22; 20(10): 79. PubMed Abstract \| Publisher Full Text\n\nRodrigues JM, Hassan M, Freiburghaus C, et al.: p53 is associated with high-risk and pinpoints TP53 missense mutations in mantle cell lymphoma. Br. J. Haematol. 2020 Dec; 191(5): 796–805. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsiao SC, Cortada IR, Colomo L, et al.: SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma. Histopathology. 2012 Oct; 61(4): 685–693. PubMed Abstract \| Publisher Full Text\n\nTroussard X, Avet-Loiseau H, Macro M, et al.: Cyclin D1 expression in patients with multiple myeloma. Hematol. J. 2000; 1(3): 181–185. PubMed Abstract \| Publisher Full Text\n\nBarista I, Romaguera JE, Cabanillas F: Mantle-cell lymphoma. Lancet Oncol. 2001 Mar; 2(3): 141–148. Erratum in: Lancet Oncol 2001 Apr;2(4):198. Erratum in: Lancet Oncol 2002 Jul;3(7):396. PubMed Abstract \| Publisher Full Text\n\nRomancik JT, Gerber DG, Zhuang T, et al.: SOHO State of the Art Updates and Next Questions: Managing Relapsed Mantle Cell Lymphoma. Clin. Lymphoma Myeloma Leuk. 2022 Aug; 22(8): 557–565. PubMed Abstract \| Publisher Full Text Publisher Full Text\n\nMonappa V, Prabhu S: Clinical and pathological characteristics of Blastoid mantle cell lymphoma: a single institution experience.March 2024. Publisher Full Text\n\nMonappa V, Prabhu S: STROBE-checklist: Clinical and pathological characteristics of Blastoid mantle cell lymphoma: A single institution experience.April 2024. Publisher Full Text" }	[ { "id": "281826", "date": "08 Jun 2024", "name": "Vijay Shankar S", "expertise": [ "Reviewer Expertise Pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors\nThe manuscript is well-written and provides valuable insights into B-MCL.\n\nHowever some of the below points to be addressed to strengthen the study a. Provide more details on how missing data and potential confounders were handled ( Eg Table 3) b. Provide clear and descriptive legends for the figure 4 c. Discussion: para3 , Line 3 sentence not grammatically 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?\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": "12052", "date": "26 Jul 2024", "name": "SWATHI PRABHU", "role": "Author Response", "response": "We authors, thank you for sparing your time in reviewing our article. We appreciate the time and efforts dedicated by you for providing feedback on our manuscript and are grateful for the insightful comments. Reviewer: Provide more details on how missing data and potential confounders were handled ( Eg Table 3) Author response: The Confounding factors with respect to IHC: As we do not have a set panel of IHC markers done for all cases [resource limited facility, patients from low socio-economic strata], usually IHC markers are requested as per individual case and differs amongst faculty; cases which were cyclin D1 & CD5 + were included in the study. We have accepted this limitation in the manuscript also. The lone case which was CD5 -ve was Cyclin D1 + (described in aggressive variants of MCL). Ki67 was done in all cases. The other markers which were done at the time of initial diagnosis are documented. P53 was the additional marker we did only on the cases (financial constraints) upon start of this study. Similarly in Table 1, only details mentioned in the EMR are documented. We had no sources to get data that were not documented. Drawbacks of retrospective nature of the study. Reviewer : Provide clear and descriptive legends for the figure 4. Author response: Thank you for this suggestion. We have updated the same in our new version of the manuscript. Reviewer : Discussion: para3 , Line 3 sentence not grammatically correct. I didn’t get what this is Author response: Thank you for pointing out this mistake. The sentence has been revised in our new version of the manuscript." } ] }, { "id": "287306", "date": "19 Jun 2024", "name": "Suma Mysore Narayana", "expertise": [ "Reviewer Expertise oncopathology with special interest in female genital tract tumors", "Breast tumors", "MGT tumors", "endocrine tumors" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA good study. I congratulate the authors for the conceptualization of this study\nI have few queries.\n\nWhat was the total number of MCL in 10years. Random 12 cases are taken as control, was there any criteria to select them. was there Cyclin D1 negative mantle lymphoma picked up with SOX11. was curious to know regarding the decision of regimens to treat MCL- was one superior over the other, BR vs R- CHOP\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "12031", "date": "18 Jul 2024", "name": "SWATHI PRABHU", "role": "Author Response", "response": "Thank you for sparing your valuable time to review our article. We appreciate time and effort in providing feedback on our manuscript and are grateful for the insightful comments. Reviewer: What was the total number of MCL in 10years. Random 12 cases are taken as control, was there any criteria to select them Author response: There were 21 cases totally, random 12 cases were selected as controls. Reviewer: Was there Cyclin D1 negative mantle lymphoma picked up with SOX11? Author response: No. SOX11 is not available in our facility. We agree that this is a potential limitation of the study. We have added this as a limitation in our manuscript. Reviewer: was curious to know regarding the decision of regimens to treat MCL- was one superior over the other, BR vs R- CHOP Response: There is not much difference in OS of patients treated with BR therapy and R-CHOP. The study involves a small cohort of patients, and the prognosis of mantle cell lymphoma (MCL) itself is poor. Hence, determining which regimen is better is difficult in this study. Clinical trials have shown that BR can be as effective as R-CHOP in terms of response rates" } ] } ]	1	https://f1000research.com/articles/13-525
https://f1000research.com/articles/13-839/v1	26 Jul 24	{ "type": "Research Article", "title": "Autonomy support in higher education: a key strategy for the well-being of university students", "authors": [ "DAVID PINEDA", "JOSE EDUARDO LOZANO-JIMENEZ", "Juan Antonio Moreno-Murcia", "DAVID PINEDA", "Juan Antonio Moreno-Murcia" ], "abstract": "Introduction Amid changing social dynamics, the world of higher education faces, among other challenges, the growing impact on the mental health of students. In this scenario, the Self-Determination Theory (SDT) highlights the important role of autonomy support as it generates positive effects on students' motivation and well-being.\n\nMethods The present study tests the predictive capacity of the teacher’s interpersonal style of autonomy support in a higher education institution, in relation to the satisfaction of basic psychological needs, autonomous motivation and depressive symptoms. A sample composed of 356 Spanish university students of which 237 were male (66.57%) from different grades and courses, aged between 17 and 57 years (M = 20.83; SD = 3.44), from middle socioeconomic strata, was used, and selected through purposive sampling.\n\nResults After the analysis of structural equations, the results showed that the teacher’s interpersonal style of autonomy support positively predicted the satisfaction of basic psychological needs and this the autonomous motivation; but negatively depressive symptoms.\n\nConclusions The model describes the possible importance of promoting the teacher’s interpersonal style of autonomy support in higher education as a protective factor for well-being and mental health. These findings highlight the importance of motivational strategies that higher education teachers must implement to promote student motivation and well-being.", "keywords": [ "motivation", "cohesion", "University students", "teaching style", "autonomy support" ], "content": "Introduction\n\nHigher education institutions face complex challenges in achieving quality and recognition (Al Abduwani, 2017; Kuoppakangas et al., 2019; Tikhonova & Raitskaya, 2018). In addition to the challenge of being recognized as centers for the generation of knowledge, innovation and technology, they have an emerging challenge: training professionals with positive psychological capacities (Siu et al., 2014), capable of advancing and concluding their training in a timely manner (Gutiérrez et al. , 2019) and with this, to respond to the productive sector, which requires competent professionals with high adaptive and coping capacities in changing and challenging environments (Moreno & Morales, 2017). These expectations find on university campuses a complex and increasingly frequent reality regarding mental health problems (Levine et al., 2022).\n\nUniversity students are considered the future of society and their well-being and mental health are a central aspect of their training (Wang et al., 2021), especially since they are a high-risk population (Chen & Lucock, 2022). Entering the university marks the beginning of a new step in the lives of young people (Teixeira et al., 2022). The transition from secondary education to university implies academic, family and personal challenges, which can become excessive pressures and worries for students, which can affect their mental health as the demand increases (Samuel & Kamenetsky, 2022), as well as their academic success, productivity and their social relationships (Dessauvagie et al., 2022).\n\nThe growing “mental health problem” in higher education impacts academic processes such as attendance, performance, engagement, and graduation (Teixeira et al., 2022). Although during the Covid-19 pandemic it faced a complex scenario (López-Núñez et al., 2021), today after having declared its end by the WHO, it continues to generate serious impacts.\n\nApproximately one in five college freshmen have experienced a mental disorder that is associated with reduced academic performance, further role impairment, and increased college dropout (Husky et al., 2022). Depression is one of the most common mental health conditions among this population. More than half of the students at public universities in the United States suffer from depression and anxiety (Limone & Toto, 2022). Its symptoms can persist over time and can significantly affect the academic aspects, relationships, and daily functioning of students (Porter, 2018).\n\nGiven this worrying scenario of mental health on university campuses, it is necessary to approach its understanding and approach (Saha et al., 2022). To this end, the Self-Determination Theory (SDT), as a model that aims to understand human behavior through the influences of the context and interpersonal perceptions (Deci & Ryan, 2008), is a key resource for the analysis of motivational processes in higher education, as a central component of student well-being (Gillet et al., 2019).\n\nThe SDT postulates that people have a natural inclination to experience a sense of choice and psychological freedom regarding their thinking and actions (Deci, 2004; Ryan & Deci, 2017) or, in other words, the tendency towards autonomous motivation. SDT starts from the fact that motivation unfolds in a continuum of processes that go from demotivation to autonomous motivation, passing through introjected motivation and integrated motivation. On this continuum, amotivation, referring to the absence of motivation, is at the opposite end of autonomous motivation, which is self-determined in nature. In the middle is extrinsic motivation, less self-determined, which oscillates between four expressions: external regulation, introjected regulation, identified regulation, and integrated regulation (Ryan & Deci, 2020). Focusing on autonomous motivation means starting from the natural tendency and makes it possible to promote it more effectively (Weidinger et al., 2016).\n\nOn the other hand, the SDT postulates the existence of three basic psychological needs (BPN): autonomy, competence and relationship with others, determinants for motivation and well-being (Šakan et al., 2020). Autonomy implies volitional aspects and the disposition of behavior based on activities oriented with the integrated sense of self and in which people perceive that they have the possibility of choice and some control over the consequences; competence is defined as the perception of feeling capable when carrying out their tasks; and the relationship with others, refers to the need of people to be meaningfully involved with others and to feel part of a group (Ryan & Deci, 2000). Satisfying these BPN is a necessary condition for the motivation “boosting” process to be successful (Ryan & Deci, 2020).\n\nBased on the above, the SDT proposes that the provision of social resources by personal networks, through an interpersonal style of autonomy support, is a predictor of BPN satisfaction (Rocchi et al., 2017; Ryan & Deci, 2000), which in turn is a predictor of autonomous motivation. Specifically, the interpersonal style of autonomy support is positioned as a central social trigger for the development of self-determined motivation in students (Zamzami & Corinne, 2019) and as a key element for academic success (Leenknecht et al., 2017), well-being and mental health (Dasinger & Gibson, 2022).\n\nWhen the BPN are satisfied, a more autonomous motivation is promoted and, consequently, positive effects are achieved (Orsini et al., 2018). Thus, although the SDT maintains that motivation is by nature a process that comes from within, it also affirms that it can be enhanced from contexts that facilitate the satisfaction of the BPN.\n\nFaced with the challenge of SDT to understand human behavior from the influences of the context (Deci & Ryan, 2008), the different motivational styles of the figures that exert some influence on other people constitute key social triggers for the satisfaction of psychological needs and, consequently, for motivation and the effects derived from it (Haerens et al., 2015).\n\nIn the educational context, the way in which teachers interact with their students constitutes a central component in SDT, insofar as the interpersonal style they adopt can influence student motivation, oscillating between a more supportive one or a more frustrating one for their BPN. When these are met, students report more motivation and engagement (Orsini et al., 2018; Patall et al., 2018) and are more likely to deeply process learning material, performing better (Jang et al., 2016), higher well-being (Gillet et al., 2019), higher educational aspirations, and lower levels of academic dropout (Ketonen et al., 2019). When teachers support students' expression and pursuit of their personal interests and goals, they become more engaged in their learning process (Bronson, 2016; Reeve & Shin, 2020).\n\nThrough their behavior, teachers can promote positive and adaptive behaviors in their students. When adopting an autonomy-supportive motivational style, students have more opportunities to take initiative and play a leadership role (Vermote et al., 2020), as they catalyze greater autonomous motivation, curiosity, and a desire to challenge (Ryan & Deci, 2000), achieving the satisfaction of their BPN and consequently developing greater autonomous motivation (Frielink et al., 2018), a key factor for academic success and well-being (Depasque & Tricomi, 2015; Tahrekhani & Sadeghian, 2015). In this way, with the satisfaction of the BPN of the students, the teachers make possible the choice, the perception of competence and the relationship with others (Soenens et al., 2018).\n\nWhen, on the contrary, teachers adopt a controlling style, based on pressure on students, which does not enhance the satisfaction of their BPN, an increase in demotivation is observed (Martinek et al., 2020) and can lead to a action oriented by fear of punishment or shame (Leenknecht et al., 2017), which is associated with a lack of commitment (Jang et al., 2016), loss of initiative and less learning (Ryan & Deci, 2000).\n\nJust as the teacher is a social trigger that can promote quality motivation, the individual characteristics of students can also promote or hinder their motivation. Recent works highlight the role of non-cognitive traits in the educational setting on motivational processes; such as the temperamental, attitudinal and motivational characteristics of the student (Fomunyam & Mnisi, 2017). These personal variables of the students interact with the teacher’s interpersonal style and must be taken into account, insofar as they can improve their own motivation, achievement and well-being (Borae & Kim, 2017; Cortez et al., 2019; Nonaillada, 2019; Seong-Lee & Chen-Hsieh, 2019).\n\nMany first-year college students experience stressors that affect their adjustment and well-being (Samuel & Kamenetsky, 2022). About a fifth suffer from some mental disorder (Dessauvagie et al., 2022). The transition to university is a stage of constant psychosocial and academic changes, in which the appearance of anxious and depressive symptoms is common (Sit et al., 2022), and is added to the aggravation of previous mental health problems (Hernández-Torrano et al., 2020), due to the developmental, academic, occupational, and social challenges they face.\n\nUniversity students present high rates of mental health problems (Limone & Toto, 2022). Depression is one of the most common in this population (Porter, 2018; Ruiz-Hernández et al., 2022), and constitutes one of the personal factors that plays a relevant role in affecting their well-being, which compromises their daily functioning and that it hinders the development of their academic career (Levine et al., 2022), to the point that up to 42.7% of students felt too depressed to function regularly (Keeler et al., 2021).\n\nFor this reason, it is particularly important to be attentive to depressive symptoms, since it is associated with other problems such as suicide, addiction and eating disorders. In particular, 20% of college students, adolescents, and young adults in the United States had suicidal tendencies for the year 2018, constituting the group with the highest percentage in the United States (Limone & Toto, 2022).\n\nDepressive symptoms among college students are higher than in the adult population and higher than their non-college peers (Chen et al., 2013). This problem even persists throughout the years of their academic training to the point that 60% of the students who indicated a mental health problem at the beginning of their career reported maintaining it two years later (Zivin et al., 2009). The most common depressive symptoms present as heightened negative affect, anhedonia, weight loss or gain, motor retardation, concentration problems, insomnia, restlessness, agitation, and feelings of worthlessness (Levine et al., 2022).\n\nSDT offers a key to the analysis, based on the fact that BPN are associated with motivational regulation and, consequently, with mental health (Teuber et al., 2021). In this direction, various studies suggest that the negative affect and frustration of BPN contribute to the development of depressive symptoms (Ryan & Deci, 2017; Šakan et al., 2020; Vansteenkiste & Ryan, 2013), especially among younger students (Shek et al., 2022). Furthermore, negative affect, along with other variables such as sensitivity to anxiety, emotional regulation, tolerance for uncertainty, or perfectionism, could be at the base of numerous psychopathological disorders, especially emotional disorders (Ferrer et al., 2018; Pineda, 2018; Pineda et al., 2018).\n\nHowever, just as the frustration of BPN is a consistent predictor of both negative affect and depressive symptoms over time (Levine et al., 2022; Šakan et al., 2020), their satisfaction, based on the implementation of a teacher’s interpersonal style that supports autonomy is a predictor of greater autonomous motivation and well-being (Gillet et al., 2019).\n\nIn this framework, although the damping effect on depression of this interpersonal style has already been investigated in primary and secondary school students (Zhang et al., 2022), it is necessary to analyze it with university students. On the other hand, although there is a very important precedent on the (negative) correlations between autonomy with respect to depression, anxiety and somatic symptoms (Sheikholeslamia & Arab-Moghaddama, 2010), it is necessary to update the findings in the present time and context.\n\nThe present study, aware of the importance of the role of the teacher in academic success (Adi Badiozaman et al., 2019; Langdon et al., 2017), and in the mental health of higher education students, aims to examine the relationships between interpersonal style of teacher autonomy support, BPN satisfaction, autonomous motivation and depressive symptoms of a group of university students, through a structural regression model.\n\n\nMethods\n\nThe study was conducted in accordance with the Declaration of Helsinki. The study was presented and approved by the ETHICS AND INTEGRITY COMMITTEE IN RESEARCH, VICE-RECTORATE FOR RESEARCH AND TRANSFER - MIGUEL HERNÁNDEZ UNIVERSITY OF ELCHE (approval number: DPS.DPS.230623. The participants agreed to be part of the study and gave their written informed consent. Everyone completed the questionnaires, willingly and anonymously to be able to respond honestly and sincerely.\n\nThe sample was made up of 356 Spanish university students, 237 of whom were boys (66.60%) from different grades and courses at a Spanish university, aged between 17 and 57 (M = 20.83; SD = 3.44). In general, coming from middle socioeconomic strata. They were selected through intentional sampling, taking into account the availability of teachers at the time of administration of the instruments. Second semester students were not included.\n\nThe present study involves the use of questionnaires that have been validated by previous studies. Below in the description of each measure is the study citation and URL. It is also included in the references section.\n\nAutonomy support. To measure the interpersonal style of autonomy support perceived by Higher Education students from their teacher, the Moreno-Murcia et al. Autonomy Support Scale (EAA) was used (Moreno-Murcia et al., 2019). It consists of 12 items (e.g. “Provides explanations that help us understand the personal utility of carrying out said activity”) and the scale begins with an introductory heading such as: “My teacher in class…”. This is rated on a Likert scale from 1 (Strongly disagree) to 5 (Strongly agree). The reliability with the present sample, measured with the alpha and omega indices have been.94 and.94 respectively.\n\nBasic psychological needs. The Spanish version of the Échelle de Satisfacción des Besoins Psychologiques in the educational context (León et al., 2011) by Gillet et al. (2008) was used. The scale was preceded by the statement “In my class…” and made up of 15 items referring to competence (e.g. “I have the feeling of doing things well”), to autonomy (e.g. “I generally feel free to express my opinions”), and to the relationship with others (e.g. “I feel good with the people with whom I relate”). The responses were established on a Likert-type scale ranging from 1 (Strongly disagree) to 5 (Strongly agree). The internal consistency in this sample, measured with the alpha and omega indices, have been .93 and .93 for competence, .92 and 92 for autonomy, and .93 and .92 for the dimension of relationship with others.\n\nAutonomous motivation. To measure student motivation, the intrinsic motivation subscales (knowledge, achievement, and stimulant) and identified motivation of the version translated and validated into Spanish by Núñez, Martín-Albo et al. (2005) from the Échelle de Motivation en Éducation (EME) (Vallerand et al., 1989) were used. The dimensions are made up of four items each (e.g. “For the pleasure I feel by expanding my knowledge on topics that interest me”). It is preceded by the phrase “Why do you study this subject?” and the responses are collected on a Likert-type scale ranging from 1 (Totally disagree) to 7 (Totally agree). The internal consistency of the scales applied in this sample, measured with the alpha and omega indices respectively, have been .92 and .92 for knowledge, .92 and .92 for achievement, .95 and .96 for the stimulating dimension and .81 and .79 for the identified motivation.\n\nDepression. To measure the depressive symptoms of the students, the PHQ-4 questionnaire (Kroenke et al., 2009) was applied. The PHQ-4 is a brief, four-item Emotional Disturbance Symptom Assessment Instrument designed for use in clinical and research settings where rapid and efficient symptom assessment is required. The PHQ-4 assesses the frequency and severity of symptoms of depression and anxiety in the last two weeks, using a response scale of 0 to 3 (0 = Never, 1 = Several days, 2 = More than half of days, 3 = Almost every day). The first two items of the PHQ-4 assess the presence and severity of depressive symptoms, including sadness and lack of interest or pleasure in activities. The last two items assess the presence and severity of anxiety symptoms, including excessive worry and difficulty relaxing. The PHQ-4 has proven to be a useful and reliable tool for the assessment of depressive and anxiety symptoms in various clinical and community settings, and its short length makes it ideal for use in settings with limited time and resources (Cano-Vindel et al., 2018). Internal consistency with the present sample has been .76 for Cronbach's alpha and .77 for McDonald's omega.\n\nThe present research has the approval of the Human Research Committee of the corresponding University, with authorization code DPS.DPS.230623 (details masked for the review process). After sharing with the Academic Department management, the teachers involved were contacted to inform them of the objective of the research and request their collaboration so that students could fill out the questionnaires in their class time. To guarantee a greater number of participants, the questionnaires were mostly administered in practical classes. The application was not made in the same subject, since none is repeated throughout the different levels of the study plan. The objective of the study and how to fill in the questionnaires were explained to them in a generic way, resolving any possible doubts that might arise during the process. In particular, the instruction was given to answer the questionnaires, not having in mind a specific subject, but rather their general experience in relation to the development of those they have taken throughout their university education. Although initially the sample was made up of more students, responses with outlier values occurred in 32 participants and it was decided to eliminate them. Once this was done, the participants agreed to be part of the study and gave their written informed consent. Everyone completed the questionnaires, willingly and anonymously to be able to respond honestly and sincerely. The time required for its completion was approximately 20 minutes.\n\n\nResults\n\nIn this study, an exploratory analysis of the data was carried out through the application of descriptive and statistical measures, to evaluate the distributions of the variables and detect possible extreme and missing values. Next, a correlation analysis was performed to assess the relationship between the variables of interest. A path analysis was then performed to examine the relationships between the variables within the proposed theoretical model, estimating the standardized regression coefficients to assess the magnitude and direction of the relationships. The model fit indices CFI, GFI, RMSEA, and SRMR were used to assess the goodness of fit of the proposed model (Byrne, 2001; Kline, 2015). All statistical analyzes were carried out using R software version 4.2.2 (R Core Team, 2022).\n\nThis section presents the results of the descriptive and correlation analysis of the data, as well as the analysis for skewness and multivariate kurtosis. The descriptive and correlation analysis of the variables has been reflected in Table 1. Regarding the results of the Mardia test, a b1p value of 17.27 was found, indicating a positive multivariate asymmetry in the data. This result is supported by the asymmetry measure, which obtained a value of 1022.09 (p < .001). In addition, the measure of asymmetry of small samples obtained a value of 1032.47 (p < .001). These results indicate that the data present a strong positive skewness in multiple dimensions. On the other hand, the value of b2p was 119.61, which suggests a positive multivariate kurtosis in the data. The kurtosis measure obtained a value of 13.8 (p < .001), which indicates a greater concentration of data around the mean, compared to the normal distribution.\n\n* p < .05.\n\n** p < .01.\n\nAn analysis was carried out with the structural equation model using the DWLS (Diagonally Weighted Least Squares) estimation method, as the most robust method to the violation of the normality assumption (Flora & Curran, 2004). The results indicate that the model adequately fits the observed data, according to the Chi-square goodness-of-fit test statistic (χ2 = 28.495, df = 8, p < .001) and other fit indices such as the global fit index (GFI = .982), the comparative fit index (CFI = .975), the root mean square error of approximation (RMSEA = .085), or the absolute mean of fit (SRMR = .080). In addition, significant relationships were found between the model variables, and the standardized regression coefficients indicate that autonomy support is positively related to autonomy satisfaction (β = .622), competence (β = .419), and interpersonal relationships (β = .648). Likewise, satisfaction with autonomy (β = .488), with competence (β = .338), and with interpersonal relationships (β = .175), predicted autonomous motivation and this, in turn, was negatively related to depression (β = -.149). These results suggest that autonomy support, autonomous motivation, as well as the satisfaction of autonomy, competence, and interpersonal relationships are important factors to consider in the prevention and treatment of depression in college students (see Figure 1).\n\nNote. The path analysis shows the association between teacher's interpersonal style of autonomy support, satisfaction of the basic needs of the student body, autonomous motivation and depressive symptoms. The coefficients presented are the standardized regression coefficients.\n\n*p<.001.\n\n\nDiscussion\n\nThe study tested a model that proposed the predictive ability of the teacher’s interpersonal style regarding depressive symptoms in college students, mediated by BPN satisfaction and autonomous motivation. All variables were positively and significantly correlated with each other, except for depressive symptoms, which were negatively correlated with BPN satisfaction and autonomous motivation. It is confirmed that the interpersonal teaching style of autonomy support positively predicts BPN satisfaction and autonomous motivation, and negatively predicts depressive symptoms in university students.\n\nThese results confirm that the teacher’s interpersonal style of autonomy support constitutes a nutrient for the satisfaction of BPN and autonomous motivation (Deci & Ryan, 2008), as long as teachers design learning opportunities that enable decision-making, the perception of competence and the construction of positive interpersonal relationships, and, consequently, are a nutrient for academic processes (Leenknecht et al., 2017) and for the well-being and mental health of university students (Dasinger & Gibson, 2022). Various studies have reported this relationship. In particular, when the BPN are satisfied, thanks to the adoption of a motivational style of autonomy support by the teacher, students report greater autonomous motivation (Frielink et al., 2018) and greater academic commitment (Orsini et al., 2018; Patall et al., 2018) expressed in better performance (Jang et al., 2016).\n\nOn the other hand, the results obtained reinforce the expectation that the motivational benefits of students are also positively related to life satisfaction, well-being and mental health, and consequently, negatively, with mental illness and depressive symptoms. Studies such as that of Gutiérrez et al. (2017) support this idea. In general, they conclude that classroom climates associated with interpersonal styles that support autonomy and cooperation are determinant for students to present high levels of satisfaction with life. Similar conclusions were reached by Lozano-Jiménez et al. (2021) in a study carried out with university students in which the predictive capacity of the teacher’s interpersonal style of autonomy support is corroborated, in relation to the satisfaction of the BPN and autonomous motivation, with satisfaction with life.\n\nIn relation to well-being and mental health Gillet et al. (2019) in a study carried out with French university students, concluded that well-being, in terms of positive affect, is positively related to BPN satisfaction. Teuber and Niewöhner (2021) also agree, pointing out, in their research with German higher education students, that BPN satisfaction is positively linked to well-being and negatively linked to depression. More recently Levine et al. (2022), concluded that BPN frustration was the only consistent predictor of both negative affect and depressive symptoms.\n\nThese results are particularly relevant, as they highlight the important role of the autonomy support style in mental health. According to Samuel and Kamenestsky (2022), students tend to use informal sources of support, such as family and friends, even with formal sources of support available through mental health services on or off campus. In this sense, teachers constitute a protective factor when in class settings they provide the necessary conditions for the empowerment of autonomous motivation as well as for the well-being of their students, especially when formal support on campus has barriers that affect access to care, either due to costs, availability and its general effectiveness (Samuel & Kamenestsky, 2022).\n\nIt is important to consider that, although the teacher’s interpersonal style of autonomy support is a resource in higher education to contribute to the well-being of students, it should also be implemented from school education, in terms of psychopathologies identified in the early years of university began before entering higher education as documented by Auerbach et al. (2019).\n\nOn the other hand, this study makes contributions of a pedagogical nature in that it highlights the importance of implementing strategies to enhance student motivation and consolidate protective factors for their mental health. Allowing decision-making, promoting active participation and teamwork, and guiding students in their study processes are key strategies to consolidate greater motivation and greater well-being (Matos et al., 2018; Cheon & Reeve, 2015). Within the framework of pedagogical processes, it is important to address the fact that most institutions have tight schedules and continuous study sequences, which affects student performance and their mental well-being. Hence, another challenge is that, in order to enhance the effects of the teacher’s interpersonal style of autonomy support, the campus environment must be adequate and protective (Limone & Toto, 2022).\n\nOne of the limitations of this study is that, since it has a correlational scope, only correlations are established between the variables treated, and although the structural equation model allows a prediction to be made, it is not possible to establish a causal relationship. Experimental studies are necessary to explain the causal relationships of the variables studied, and others in which the sample is random and equally distributed by gender. In addition to the issue of scope, the type of cross-sectional design adopted does not allow an analysis to proceed over a longer timeline. This makes it necessary for subsequent studies to measure the evolution of the variables in various time cuts. Similarly, the proposed model is the one that presented the best fit, but due to the problem of equivalent models presented by the structural equations technique (Hershberger, 2006), it is assumed that the proposed model would be only one of the possible ones.\n\nAs practical implications, in a higher education scenario, the consideration of certain personal variables of the student related to self-regulation should be elements that serve as a basis to complement and guide effective pedagogical practices based on promoting autonomy support and strengthening success-oriented academic and the promotion of students’ mental health. This represents a challenge, since according to Ryan and Deci (2020) conventional relationship styles are installed under the protection of institutional models and educational policies conventionally focused on control practices. Finally, results suggest promoting a much broader and more diverse university teacher training, focused not only on deep specialization and expertise in an academic area and on research, but also on mastering effective teaching-learning strategies (Oriol-Granado et al., 2017).\n\nAs a conclusion, in the context of higher education, the teacher’s interpersonal style of autonomy support facilitates the satisfaction of basic psychological needs and, consequently, enhances autonomous motivation, which enables students to perceive depressive symptoms with less intensity.", "appendix": "Data availability\n\nAll data supporting this research are available in a public repository, in order to promote the integrity, discovery and reuse of this research. Data are available in:\n\nDates of PIIE. 24542071\n\nMoreno-Murcia, Juan Antonio. Dates of PIIE. figshare. Dataset.\n\nDOI 10.17605/OSF.IO/GM4JT\n\nhttps://osf.io/gm4jt/?view_only=ead887f5ba2345fc96ed95d1725957d7\n\nData is available under the terms of the Creativa Commons - CC BY 4.0 DEED\n\nAttribution 4.0 International\n\n\nReferences\n\nAdi Badiozaman I, Leong H, Jikus O: Investigating student engagement in Malaysian higher education: a self-determination theory approach. J. Furth. High. Educ. 2019; 44: 1364–1378. Publisher Full Text\n\nAl Abduwani T: Global challenges in higher education: a gulf perspective. Asian Journal of Social Sciences, Arts and Humanities. 2017; 5(1): 46–53.\n\nAuerbach RP, Mortier P, Bruffaerts R, et al.: Mental disorder comorbidity and suicidal thoughts and behaviors in the World Health Organization World Mental Health Surveys International College Student initiative. Int. J. Methods Psychiatr. Res. 2019; 28(2): e1752. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorae J, Kim J: Grit, Basic Needs Satisfaction, and Subjective Well-Being. J. Individ. Differ. 2017; 38(1): 29–35. Publisher Full Text\n\nBronson S: Autonomy support environment and autonomous motivation on nursing student academic performance: An exploratory analysis. Nurse Educ. Today. 2016; 44: 103–108. PubMed Abstract \| Publisher Full Text\n\nByrne BM: Structural equation modeling with AMOS, EQS, and LISREL: Comparative approaches to testing for the factorial validity of a measuring instrument. Int. J. Test. 2001; 1(1): 55–86. Publisher Full Text\n\nCano-Vindel A, Muñoz-Navarro R, Medrano LA, et al.: A computerized version of the Patient Health Questionnaire-4 as an ultra-brief screening tool to detect emotional disorders in primary care. J. Affect. Disord. 2018; 234: 247–255. PubMed Abstract \| Publisher Full Text\n\nChen T, Lucock M: The mental health of university students during the COVID-19 pandemic: An online survey in the UK. PLoS One. 2022; 17(1): e0262562–e0262517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen L, Wang L, Qiu XH, et al.: Depression among Chinese University Students: Prevalence and socio-demographic correlates. PLoS One. 2013; 8: 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheon S, Reeve J: A classroom-based intervention to help teachers decrease students’ amotivation. Contemp. Educ. Psychol. 2015; 40: 99–111. Publisher Full Text\n\nCortez A, Winer L, Kim Y, et al.: Predictors of medical student success on the surgery clerkship. Am. J. Surg. 2019; 217: 169–174. PubMed Abstract \| Publisher Full Text\n\nCumming G: The new statistics: Why and how. Psychol. Sci. 2014; 25(1): 7–29. Publisher Full Text\n\nDasinger T, Gibson D: Perceptions of mental health and need satisfaction/frustration among rural university students. J. Am. Coll. Heal. 2022; 72: 253–260. PubMed Abstract \| Publisher Full Text\n\nDeci E: Intrinsic Motivation and Self-Determination.Spielberger C, editor. Encyclopedia of Applied Psychology. Rochester: University of Rochester; 2004; pp. 437–448. Publisher Full Text\n\nDeci E, Ryan R: Self-Determination Theory: A Macrotheory of Human Motivation, Development, and Health. Can. Psychol. 2008; 49(3): 182–185. Publisher Full Text\n\nDepasque S, Tricomi E: Effects of intrinsic motivation on feedback processing during learning. NeuroImage. 2015; 119: 175–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDessauvagie A, Dang H, Nguyen T, et al.: Mental Health of University Students in Southeastern Asia: A Systematic Review. Asia Pac. J. Public Health. 2022; 34(2-3): 172–181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerrer L, Martín-Vivar M, Pineda D, et al.: Relación de la ansiedad y la depresión en adolescentes con dos mecanismos transdiagnósticos: el perfeccionismo y la rumiación [Relationship of anxiety and depression in adolescents on two transdiagnostic mechanisms: perfectionism and rumination]. Behav. Psychol. 2018; 26(1): 55–74.\n\nFlora DB, Curran PJ: An empirical evaluation of alternative methods of estimation for confirmatory factor analysis with ordinal data. Psychol. Methods. 2004; 9(4): 466–491. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFomunyam KG, Mnisi T: Temperament as a Determinant of Success in Formative Assessment in Engineering Education. Int. J. Appl. Eng. Res. 2017; 12(14): 4152–4161.\n\nFrielink N, Schuengel C, Embregts PJCM: Autonomy Support, Need Satisfaction, and Motivation for Support among adults with intellectual disability: Testing a Self-Determination Theory Model. Am. J. Intellect. Dev. Disabil. 2018; 123(1): 33–49. PubMed Abstract \| Publisher Full Text\n\nGillet N, Morin A, Huyghebaert T, et al.: University students’ need satisfaction trajectories: A growth mixture analysis. Learn. Instr. 2019; 60: 275–285. Publisher Full Text\n\nGillet N, Rosnet E, y Vallerand, R. J.: Developpement d'une echelle de satisfaction des besoins fondamentaux en contexte sportif. Can. J. Behav. Sci. 2008; 40(4):230–237. Publisher Full Text\n\nGutiérrez JA, Mondragón V, Santacruz LC: Expectativas, necesidades y tendencias de la formación en educación superior en Colombia en pregrado y posgrado: entre la deserción-perfil y vocación profesional [Expectations, needs and trends in undergraduate and graduate higher education in Colombia: between attrition-profile and vocational vocation]. Revista Universidad y Empresa. 2019; 21(37): 313–345. Publisher Full Text\n\nGutiérrez M, Tomás J, Calatayud P: Influence of the motivational climate in Physical Education on learning goals and life satisfaction. Retos. 2017; 31: 157–163. Publisher Full Text\n\nHaerens L, Aelterman N, Vansteenkiste M, et al.: Do perceived autonomy-supportive and controlling teaching relate to physical education students’ motivational experiences through unique pathways? Distinguishing between the bright and dark side of motivation. Psychol. Sport Exerc. 2015; 16(3): 26–36. Publisher Full Text\n\nHernández-Torrano D, Ibrayeva L, Sparks J, et al.: Mental Health and Well-Being of University Students: A Bibliometric Mapping of the Literature. Front. Psychol. 2020; 11: 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHershberger S: The problem of equivalent structural models.Hancock GR, Mueller RO, editors. Structural equation modeling: a second course. Information Age Publishing; 2006; pp. 13–42.\n\nHusky M, Sadikova E, Lee S, et al.: Childhood adversities and mental disorders in first-year college students: results from the World Mental Health International College Student Initiative. Psychol. Med. 2022; 53(7): 2963–2973. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJang H, Reeve J, Halusic M: A new autonomy-supportive way of teaching that increases conceptual learning: Teaching in students’ preferred ways. J. Exp. Educ. 2016; 84(4): 686–701. Publisher Full Text\n\nKeeler L, Skidmore B, Leenstra T, et al.: Treating University Students’ Depression using Physical Activity with Peers: Two Field-Based Quasi-Experiments Grounded in the Self-Determination Theory. J. Coll. Stud. Psychother. 2021; 35(3): 205–223. Publisher Full Text\n\nKetonen E, Malmberg L, Salmela-Aro K, et al.: The role of study engagement in university students’ daily experiences: A multilevel test of moderation. Learn. Individ. Differ. 2019; 69: 196–205. Publisher Full Text\n\nKline R: Principles and practice of structural equation modeling. Guilford Publications; 2015.\n\nKroenke K, Spitzer RL, Williams JB, et al.: An ultra-brief screening scale for anxiety and depression: The PHQ–4. Psychosomatics. 2009; 50: 613–621. PubMed Abstract \| Publisher Full Text\n\nKuoppakangas P, Suomi K, Stenvall J, et al.: Revisiting the five problems of public sector organisations and reputation management—The perspective of higher education practitioners and ex-academics. Int. Rev. Public Nonprofit Mark. 2019; 16: 147–171. Publisher Full Text\n\nLangdon J, Schlote R, Melton B, et al.: Effectiveness of a need supportive teaching training program on the developmental change process of graduate teaching assistants' created moivational climate. Psychol. Sport Exerc. 2017; 28: 11–23. Publisher Full Text\n\nLeenknecht M, Wijnia L, Loyens S, et al.: Need-supportive teaching in higher education: Configurations of autonomy support, structure, and involvement. Teach. Teach. Educ. 2017; 68: 134–142. Publisher Full Text\n\nLeón J, Domínguez E, Núñez JL, et al.: Translation and validation of the Spanish version of the Échelle de Satisfaction des Besoins Psychologiques in academic context. Anales de Psicología. 2011; 27(2): 405–411.\n\nLevine SL, Brabander CJ, Moore AM, et al.: Unhappy or unsatisfied: distinguishing the role of negative affect and need frustration in depressive symptoms over the academic year and during the COVID-19 pandemic. Motiv. Emot. 2022; 46(1): 126–136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLimone P, Toto G: Factors That Predispose Undergraduates to Mental Issues: A Cumulative Literature Review for Future Research Perspectives. Front. Public Health. 2022; 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLópez-Núñez A, Piqueras JA, Falcó R, et al.: The Mediating Role of Psychological Strengths Between Academic/Work Interference and Mental Health during COVID-19 Lockdown. Acción Psicológica. 2021; 18(1): 135–150. Publisher Full Text\n\nLozano-Jiménez JE, Huéscar E, Moreno-Murcia JA: From Autonomy Support and Grit to Satisfaction With Life Through Self-Determined Motivation and Group Cohesion in Higher Education. Front. Psychol. 2021; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartinek D, Zumbach J, Carmignola M: The impact of perceived autonomy support and autonomy orientation on orientations towards teaching and self-regulation at university. Int. J. Educ. Res. 2020; 102: 101574–101578. Publisher Full Text\n\nMatos L, Reeve J, Herrera D, et al.: Students' Agentic Engagement Predicts Longitudinal Increases in Perceived Autonomy-Supportive Teaching: The Squeaky Wheel Gets the Grease. J. Exp. Educ. 2018; 86(4): 579–596. Publisher Full Text\n\nMoreno R, Morales S: Las competencias socio-personales para la inserción sociolaboral de jóvenes en los programas propios de educación social [Socio-personal competencies for the socio-labor insertion of young people in social education programs]. Revista Española de Orientación y Psicopedagogía. 2017; 28(1): 33–50. Publisher Full Text\n\nMoreno-Murcia JA, Huéscar E, Pintado R, et al.: Diseño y validación de la Escala de Apoyo a la Autonomía en educación superior: Relación con la competencia laboral del discente [Design and validation of the Autonomy Support Scale in higher education: Relationship with student labor competence]. Revista Española de Orientación y Psicopedagogía. 2019; 30(1): 116–130. Publisher Full Text\n\nNonaillada J: Applying Self-Determination Theory (SDT) to Faculty Engagement for Curriculum Development. J. Fac. Dev. 2019; 33(3): 103–108.\n\nNúñez J, Martín-Albo J, Navarro J: Validación de la versión española de la Échelle de Motivation en Éducation [Validation of the Spanish version of the Échelle de Motivation en Éducation]. Psicothema. 2005; 17(2): 344–349.\n\nOriol-Granado X, Mendoza-Lira M, Covarrubias-Apablaza C, et al.: Positive Emotions, Autonomy Support and Academic Performance of University Students: The Mediating Role of Academic Engagement and Self-efficacy. Revista de Psicodidáctica. 2017; 22(1): 45–53. Publisher Full Text\n\nOrsini C, Binnie V, Tricio J: Motivational profiles and their relationships with basic psychological needs, academic performance, study strategies, self-esteem, and vitality in dental students in Chile. J. Educ. Eval. Health Prof. 2018; 15(11): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatall E, Steingut R, Vasquez A, et al.: Daily autonomy supporting or thwarting and students’ motivation and engagement in the high school science classroom. J. Educ. Psychol. 2018; 110(2): 269–288. Publisher Full Text\n\nPineda D: Procesos transdiagnósticos asociados a los síntomas de los trastornos de ansiedad y depresivos [Doctoral dissertation, UNED. Universidad Nacional de Educación a Distancia].2018. Reference Source\n\nPineda D, Valiente RM, Chorot P, et al.: Invarianza factorial y temporal del Cuestionario de Regulación Emocional (ERQ) [Factorial and temporal invariance of a Spanish version of the Emotional Regulation Questionnaire (ERQ)]. Revista de Psicopatología y Psicología Clínica. 2018; 23(2): 109–120. Publisher Full Text\n\nPorter S: A descriptive study of post-secondary student mental health crises. Coll. Q. 2018; 21: 3.\n\nR Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2022. Reference Source\n\nReeve J, Shin S: How teachers can support students’ agentic engagement. Theory Pract. 2020; 59(2):150–161. Publisher Full Text\n\nRocchi M, Pelletier L, Cheung S, et al.: Assessing need-supportive and need-thwarting interpersonal behaviours: The Interpersonal Behaviours Questionnaire (IBQ). Personal. Individ. Differ. 2017; 104: 423–433. Publisher Full Text\n\nRuiz-Hernández JA, Guillén Á, Pina D, et al.: Mental Health and Healthy Habits in University Students: A Comparative Associative Study. Eur. J. Investig. Health Psychol. Educ. 2022; 12: 114–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyan RM, Deci EL: Self-determination theory and the facilitation of intrinsic motivation, social development, and well-being. Am. Psychol. 2000; 55(1): 68–78. PubMed Abstract \| Publisher Full Text\n\nRyan RM, Deci EL: Self-Determination Theory: Basic psychological needs in motivation, development, and wellness. Guilford; 2017. Publisher Full Text\n\nRyan RM, Deci EL: Intrinsic and extrinsic motivation from a self-determination theory perspective: Definitions, theory, practices, and future directions. Contemp. Educ. Psychol. 2020; 61: 101811–101860. Publisher Full Text\n\nSaha K, Yousuf A, Boyd RL, et al.: Social Media Discussions Predict Mental Health Consultations on College Campuses. Sci. Rep. 2022; 12: 111–123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nŠakan D, Žuljević D, Rokvić N: The Role of Basic Psychological Needs in Well-Being During the COVID-19 Outbreak: A Self-Determination Theory Perspective. Front. Public Health. 2020; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamuel R, Kamenetsky SB: Help-Seeking Preferences and Factors Associated with Attitudes Toward Seeking Mental Health Services Among First-Year Undergraduates. Can. J. High. Educ. 2022; 52(1): 30–50. Publisher Full Text\n\nSeong-Lee J, Chen-Hsieh J: Affective variables and willingness to communicate of EFL learners in in-class, out-of-class, and digital contexts. System. 2019; 82: 63–73. Publisher Full Text\n\nSheikholeslamia R, Arab-Moghaddama N: Relations of autonomy and adjustment in Iranian college students: a cross-culture study of self-determination theory. Soc. Behav. Sci. 2010; 5: 1831–1835. Publisher Full Text\n\nShek D, Dou D, Zhu X, et al.: Need Satisfaction and Depressive Symptoms Among University Students in Hong Kong During the COVID-19 Pandemic: Moderating Effects of Positive Youth Development Attributes. Front. Psych. 2022; 13: 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSit H, Hong I, Burchert S, et al.: A Feasibility Study of the WHO Digital Mental Health Intervention Step-by-Step to Address Depression Among Chinese Young Adults. Front. Psych. 2022; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiu O, Bakker A, Jiang X: Psychological capital among university students: Relationships with study engagement and intrinsic motivation. J. Happiness Stud. 2014; 15: 979–994. Publisher Full Text\n\nSoenens B, Vansteenkiste M, Van Petegem S, et al.: How to solve the conundrum of adolescent autonomy? On the importance of distinguishing between independence and volitional functioning.Soenens B, Vansteenkiste M, Van Petegem S , editors. Autonomy in adolescent development: Towards conceptual clarity. Routledge; 2018; pp. 1–32.\n\nTahrekhani M, Sadeghian Z: Intrinsic Motivation Comparative Investigation between Nursery, Midwifery, and Medicine Students During Internship in Iran. Procedia. Soc. Behav. Sci. 2015; 185: 185–189. Publisher Full Text\n\nTeixeira S, Ferré-Grau C, Canut TL, et al.: Positive Mental Health in University Students and Its Relations with Psychological Vulnerability, Mental Health Literacy, and Sociodemographic Characteristics: A Descriptive Correlational Study. Int. J. Environ. Res. Public Health. 2022; 19(6): 3185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeuber Z, Jia H, Niewöhner T: Satisfying students’ psychological needs during the COVID-19 outbreak in German higher education institutions [Original Research]. Front. Educ. 2021; 6. Publisher Full Text\n\nTikhonova E, Raitskaya L: An overview of trends and challenges in higher education on the worldwide research agenda. Int. J. Lang. Educ. 2018; 4(4): 4–7. Publisher Full Text\n\nVallerand R, Blais M, Brière N, et al.: Construction et validation de l'échelle de motivation en éducation (EME) [Construction and validation of the Motivation toward Education Scale]. Can. J. Behav. Sci. 1989; 21(3): 323–349. Publisher Full Text\n\nVansteenkiste M, Ryan RM: On psychological growth and vulnerability: Basic psychological need satisfaction and need frustration as a unifying principle. J. Psychother. Integr. 2013; 23: 263–280. Publisher Full Text\n\nVermote B, Aelterman N, Beyers W, et al.: The role of teachers’ motivation and mindsets in predicting a (de) motivating teaching style in higher education: a circumplex approach. Motiv. Emot. 2020; 44(2): 270–294. Publisher Full Text\n\nWang C, Wen W, Zhang H, et al.: Anxiety, depression, and stress prevalence among college students during the COVID-19 pandemic: A systematic review and meta-analysis. J. Am. Coll. Heal. 2021; 71: 2123–2130. PubMed Abstract \| Publisher Full Text\n\nWeidinger A, Spinath B, Steinmayr R: Why does intrinsic motivation decline following negative feedback? The mediating role of ability self-concept and its moderation by goal orientations. Learn. Individ. Differ. 2016; 47: 117–128. Publisher Full Text\n\nZamzami Z, Corinne J: Exploring students’ competence, autonomy and relatedness in the flipped classroom pedagogical model. J. Furth. High. Educ. 2019; 43(1): 1–12. Publisher Full Text\n\nZhang D, Jin B, Cui Y: Do Teacher Autonomy Support and Teacher–Student Relationships Influence Students’ Depression? A 3-Year Longitudinal Study. Sch. Ment. Heal. 2022; 14: 110–124. Publisher Full Text\n\nZivin K, Eisenberg D, Gollust SE, et al.: Persistence of mental health problems and needs in a college student population. J. Affect. Disord. 2009; 117(3): 180–185. PubMed Abstract \| Publisher Full Text" }	[ { "id": "314915", "date": "03 Oct 2024", "name": "Domenico Monacis", "expertise": [ "Reviewer Expertise physical activity", "sport science", "sports 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\nI first would like to congratulate the author for this interesting study, about autonomy support in higher education. It’s a considerable effort from the researchers and the constructs involved. It’s well written and follow the IMRAD design and describes adequately the content of the research protocol. It’s well written and follow the IMRAD design and describes adequately the content of the research protocol. The background of the study is clear and points out the need to further research in this field. The methods and findings are clear, and well justified as well as data analysis. I would suggest specifying how participants have been recruited in a more detailed way. Moreover, the results and discussion are well written, and present and discuss the main results of the research in adequate manner, clearly presenting the main outcomes of the research, and discussing them accordingly. I would like to congratulate the author for the enormous effort in doing this research and acknowledge the contribution for the understanding of the topic. We would suggest clarification of the contents above, for easier understanding, but besides that, in my opinion, should be accepted. Best compliments\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "308755", "date": "25 Nov 2024", "name": "Ana Velasquez", "expertise": [ "Reviewer Expertise Social and emotional development", "Classroom climate" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examined the relationship among several motivational variables and mental health (particularly depression), in a group of 356 Spanish university students. It was found that university teachers’ autonomy support predicted students’ reports of their satisfaction of basic psychological needs. The satisfaction of these needs was also a predictor of the students’ autonomous motivation, which, in turn, was negatively related with depressive symptoms. All of this was tested through structural equation models, with cross-sectional data.\nThe study provides support for self-determination theory, as it shows the relevance that teachers’ autonomy support has for students’ motivation and wellbeing. The paper presents and organized rationale, which is well supported by pertinent and recent literature. The methodological design is sound enough to test for the proposed hypothesis, and the limitations stated info the discussion section acknowledge that no causal inferences may be made.\nIn the introduction, second paragraph, the authors need to further explain that statement that university students are a high-risk population.\nIn the discussion section, the authors state that they “tested a model that proposed the predictive ability of the teacher’s interpersonal style regarding depressive symptoms in college students, mediated by BPN satisfaction and autonomous motivation.” The interpretation about the mediation effect should be restated, as the analyses do not include all the steps needed to prove a mediation hypothesis. Neither the direct effects between the predictor (i.e., teachers’ autonomy support) and the dependent variables (i.e., depressive symptoms) were tested, nor the change in those effects once the meditators are included in the model were analyzed.\nAs for the source data, it was provided, but a data dictionary should also be provided so that any researcher can calculate de variables an reproduce the results.\nFinally, there are some writing mistakes that should be corrected. For example: -Abstract/Results: \"basic psychological needs and this the autonomous\" -Introduction, Personal triggers… section, third paragraph: \" which compromises their daily functioning and that it hinders” -Methods, Ethics, first paragraph: a closing parenthesis is missing for “(approval number…”\nOnce these issues have been revised, I think the paper will be eligible of indexing in this outlet.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-839
https://f1000research.com/articles/13-838/v1	26 Jul 24	{ "type": "Research Article", "title": "Immediate effects of Thai foot massage on renal blood flow, psychological stress, and heart rate variability in community-dwelling older adults: a randomized controlled trial", "authors": [ "Yada Thadanatthaphak", "Jaturat Kanpittaya", "Wittawat Takong", "Sutin Chanaboon", "Kukiat Tudpor", "Yada Thadanatthaphak", "Jaturat Kanpittaya", "Wittawat Takong", "Sutin Chanaboon" ], "abstract": "Background Renal blood flow (RBF) is regulated by an autonomic nervous system and is reduced in older adults. Massage has been previously found to increase blood flow.\n\nObjective This two-armed double-blind, randomized controlled trial aimed to investigate the immediate effects of Thai foot massage (TFM) on RBF, psychological stress, and heart rate variability (HRV) in older adult persons.\n\nMaterial and Methods The 26 healthy older adult volunteers were recruited and randomly assigned to the TFM group (13 persons) and the control group (13 persons). The TFM group received a 15-minute Thai foot massage, and the control group received a 15-minute bed rest. Primary outcomes – RBF parameters [peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI), volumetric arterial blood flow (VF)] and secondary outcomes – HRV parameters [standard deviation of the normal-to-normal intervals (SDNN), root mean square of successive differences (RMSSD), high frequency (HF), low frequency (LF), and low frequency per high frequency (LF/HF)] were measured after each intervention.\n\nResults Results showed that the VF significantly increased after TFM (P < 0.05) but not in control. Meanwhile, the stress index significantly reduced after TFM (P < 0.05). SDNN and RMSSD, the proxies of parasympathetic activity, also significantly increased in the TFM group (p < 0.05). Only RMSSD was significantly enhanced in the control group. No side effects were observed.\n\nConclusion The TFM could increase RBF and alleviate psychological stress through parasympathetic activity actuation. Therefore, this intervention might improve RBF and relieve stress in the older population. Further study should be carried out on a larger population.", "keywords": [ "Foot massage", "renal blood flow", "parasympathetic activity", "heart rate variability", "autonomic nervous system." ], "content": "Introduction\n\nRenal blood flow (RBF) plays a crucial role in maintaining an oxygen supply capable of meeting the demands of renal function and driving glomerular filtration by supplying sufficient capillary pressure.1 Aging affects every tissue and organ in the human body, including the kidneys. The main features of the structural and functional changes that come with kidney aging are a shrinking in size, a decline in the number of functioning glomeruli, and abnormalities in the blood vessels.2 It was discovered that the RBF decreased by around 10% every decade beyond the age of 40, with an average reduction of approximately -85 mL/min each decade across various investigations.1\n\nFactors influencing the RBF include circadian cycle, food intake, smoking, alcohol consumption, medications, physical exercise, and mental stress. According to a reference work entry by Solomon, mental stress or psychological stress is defined as “a form of stress that occurs because of how events in one’s external or internal environment are perceived, resulting in the psychological experience of distress and anxiety3”. Psychological stress can be determined by heart rate variability (HRV), which provides readouts of both sympathetic and parasympathetic variables as well as the stress index.4 It has been evidenced that psychological stress reduces the RBP through sympathoadrenal excitation-mediated vasoconstriction.1,5,6 Thus, the factors altering psychological stress are implied to modify the RBF.\n\nThai massage is a distinct form of therapy that involves deep tissue massage with the thumbs, frequently paired with post-session muscular stretching. Benefits of the massage include improved skin warmth and blood flow, decreased anxiety, decreased depression, decreased sympathetic activity, increased parasympathetic activity, and reduced cortisol and salivary α-amylase levels, indicators of psychological stress.7 The Thai foot massage (TFM) has recently increased RBF in young people.8 Therefore, this study aimed to investigate the immediate effects of Thai foot massage on RBF and HRV in older adults.\n\n\nMethods\n\nThis study was a two-armed double-blind, randomized controlled trial (RCT). Physical examinations, including blood pressure and urine tests, were performed at Suddhavej Hospital, Faculty of Medicine, Mahasarakham University, to measure the basal health status of the volunteers. The experiments and the measurement of renal blood flow and HRV were conducted in the ultrasound room, where temperature and humidity were set at 25 °C and 60-70%, respectively, at Srinagarind Hospital, Khon Kaen University, a tertiary care university medical center. Written informed consent forms were obtained from all patients. The eligibility criteria for participants were age 60 and older and healthy (not diagnosed by physicians with chronic diseases). Forty older adult volunteers were recruited. Seven volunteers were excluded due to kidney stones, while the other seven refused to participate in the study for personal reasons. The study was carried out between June and December 2017. Repositories of the data can be found at https://osf.io/yrf3j/.9 The research report complies with the Consolidated Standards of Reporting Trials (CONSORT) (Figure 1). A copy of the original trial protocol, a completed CONSORT checklist, and a flow diagram can be found in the Extended data.9 The study has been approved by the Thai Clinical Trials Registry (TCTR), which became the primary registry of WHO on August 7, 2013 (https://www.thaiclinicaltrials.org/). Our TCTR identification number is TCTR20240526002 (https://www.thaiclinicaltrials.org/show/TCTR20240526002). There were no changes to methods after trial commencement.\n\nAll procedures have been registered to the Thai Clinical Trials Registry (TCTR20240526002), approved by the Institutional Review Board (Name: Ethical Review Committee for Human Research, Mahasarakham University, Thailand) with the approval number 014/2559, 29th May 2017 and carried out following the ethical principles in the Declaration of Helsinki. All participants signed a consent form stating that data are blinded and exclusively available for professional research staff.\n\nBefore the intervention, the participants were instructed to acclimate themselves by spending 15 minutes in the supine lying posture in bed. Renal hemodynamics (RBF) were measured for 2.5 minutes after the ultra-short-term HRV values were collected. Each participant underwent a blinded, simple random sampling by drawing the assigned number from an indistinguishable container. Y.T. generated the random allocation sequence, while K.T. enrolled and assigned participants to interventions. The experimental group received a 15-minute Thai foot massage using moderate thumb press along three lines on both feet in a supine lying position as previously described10 (Figure 2). The control group was mandated to rest for 15 minutes. The HRV measurements and RBF were reevaluated right away following the intervention by J.K. and W. T, blinded from the group assignment.\n\nAs previously mentioned,11 photoplethysmography (PPG) on the participant’s left index fingertips coupled to the computer was used to measure the stress index and HRV parameters for 2.5 minutes by a physical therapist using uBioMacpa software version 1.0 (BioSense Creative Co., Ltd, Korea) (http://www.ubionet.com) (Figure 3). This software is proprietary and a free alternative is the Kubios HRV Scientific Lite. The stress index scores were divided into five groups: chronic stress (≥ 60), accumulative stress (45–59), primary stress (35–44), temporary stress (25–34), and no stress (< 25). The parasympathetic proxies uBioMacpa (high frequency (lnHF), a standard deviation of all normal R-R intervals (SDNN), and the square root of the mean of the squared successive differences in R-R intervals (RMSSD) are among the sympathetic proxies (low frequency (lnLF) and low/high-frequency ratio (LF/HF ratio).\n\nA, photoplethysmography; B, uBioMacpa software.\n\nThe RBF parameters, including peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI), and volumetric arterial blood flow (VF), were measured in a prone lying position with a linear 4.4-13 MHz probe of Doppler ultrasound (ProSound F75 continuous-wave ultrasound, Hitachi-Aloka). The ultrasound probe was placed between the 12th thoracic vertebra and the 2nd lumbar vertebra, 5 cm lateral to the spine, to measure blood flow of the left renal artery. The RI was calculated from PSV-EDV/PSV.12 The VF is equal to cross-sectional area (A) x time-averaged velocity (TAV). As previously described, the A and TAV can be calculated as π x radius.2,13 These parameters were measured twice, and the mean of each parameter was recorded. RBF was measured immediately after the measurement of HRV parameters by the radiologists (Figure 4).\n\nWith an effect size of 1.2,14 α-error level (two-sided)=0.05, and 1-β error level=0.95, the G*power algorithm was used to determine the sample size for each group, yielding n=12/group.\n\nThe data were expressed as mean±standard error of the mean (SEM). Shapiro–Wilk test was used to verify normal distribution. Paired t-test was used to compare the outcome variables between before and after experiments within a group. An independent t-test was used to compare outcome variables between groups. Statistical significance was set at the P<0.05.\n\n\nResults\n\nThe remaining twenty-six volunteers were divided into two groups, the TFM group (13 persons; female=11, male=2), with an average age of 68.15±1.52 years old. They received a 15-minute Thai foot massage using moderate thumb press along three lines on both feet in a supine lying position (Figure 2). The control group (13 persons; female=10, male=3) had an average age of 65.00±1.10 years old. They received a 15-minute bed rest in the same position. The volunteers were advised to refrain from eating, drinking alcohol, smoking, and consuming caffeine for at least 2 hours before participating in this study. Their basal renal function (urinary creatinine excretion rate) and predicted muscle mass were not significantly different.\n\nWithin-group comparison of the means between before and immediately after experiments demonstrated that VF was significantly increased only in the TFM group (p=0.012) but not in the control group. PSV, EDV, and RI were not changed in both groups. No side effects were observed.\n\nData values are expressed as mean±SEM. Thai foot massage (TFM) values are compared to control values with independent t-tests.\n\nData values are expressed as mean±SEM. Pre-intervention values are compared to post-intervention values with paired t-tests. RBF parameters include volumetric arterial blood flow (VF), peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI).\n\nSDNN, RMSSD, and HF were significantly increased in the TFM group (p=0.000, p=0.020, p=0.019, respectively). RMSSD was significantly increased in the C group (p=0.032). However, LF and LF/HF ratio were not changed in the TFM group, and SDNN, HF, LF, and LF/HF ratio were not altered in the C group (Table 1 and Table 2). The between-group comparison showed no difference between the groups in all parameters.\n\nHRV parameters include stress index, standard deviation of the normal-to-normal intervals (SDNN), root mean square of successive differences (RMSSD), high frequency (HF), low frequency (LF), and low frequency per high frequency (LF/HF) ratio (Table 3).\n\n\nDiscussion\n\nThis study demonstrated that TFM increased renal blood flow and reduced psychological stress through parasympathetic nervous system activation in older adults. Our statement is based on the following findings. First, the TFM increased the VF by approximately 20%. Secondly, the TFM significantly reduced the stress index. Lastly, the TFM increased the SDNN and RMSSD, the proxies of the parasympathetic activity.\n\nDoppler ultrasonography is a widely used non-invasive technique for examining the morphology and function of the intraparenchymal vascularity of the kidney.15 The VF can be estimated using the Doppler approach, which involves multiplying the flow velocity by the cross-sectional lumen diameter at that exact moment in time.16 Using the magnetic resonance imaging (MRI) method, the renal artery VF reportedly ranged from 200 to 1200 mL/min.17 The VF values in the present study (400-500 mL/min) are within the normal limits. Alternatively, the blood flow velocity can also be measured as an index of vascular function. A previous study by Iwamoto and colleagues demonstrated that a two-minute friction massage around the halfway point of the gastrocnemius muscle’s medial and lateral heads increased the popliteal venous blood flow velocity.18 Our present finding of the increment of approximately 20% of the VF after the TFM aligns with our previous report on the stimulatory effect of active ankle movements on the VF.19 The mechanism of the massage-induced VF increase is unclear, but the previous study showed that passive muscle movement could increase cerebral blood oxygenation in older persons.20 Therefore, the TFM probably improves the VF of the renal artery in the same way. Previously, the 8-min reflexology-based foot massage was reported to reduce the RI of the renal artery. However, the RI and other RBF parameters (PSV and EDV) were not altered by the TFM in the present study.\n\nWe found that the PSV and EDV in the renal artery are approximately 50-55 and 13-16 cm/s, respectively. These values are lower than the typical ranges of previously reported PSV (60-100 cm/s) and EDV (20-50 cm/s).21,22 Previous studies showed that the PSV and EDV in older adults were approximately 60-70 and 19-21 cm/s.23 Consequently, our lower PSV and EDV findings resulted in elevated RI (> 0.7).24 The exalted RI might be explained by the averaged participant’s high blood pressure in the present study, as shown in over two-thirds of hypertensive patients without renal artery stenosis.25 Notably, those with RI ≥ 0.70 had a lower estimated glomerular filtration rate (eGFR) than those with RI < 0.70.25\n\nAs mentioned, the RBF is closely related to psychological stress. The psychological stress leads to renal vasoconstriction, discernibly due to increased sympathetic nervous activity in the kidneys.26 Moreover, a higher degree of psychological stress has been linked with a greater likelihood of an accelerated eGFR decline.27 Based on our present finding of the TFM-reducing stress index, it is tempting to further inspect the relationship between the RBF, psychological stress, and autonomic nerve activities in a chronic kidney disease subpopulation group.\n\nOur findings of TFM-induced SDNN and RMSSD indicate an increase in parasympathetic activity. These observations accord with the previous report on the parasympathetic nervous system stimulation by foot reflexology, which also causes the release of endogenous substances. According to this idea, localized enzymatic reactions in receptive fields and skin-to-skin contact raise localized skin temperatures, enhancing physical function and blood flow.28,29 It has also been shown that foot massage increases vagal activity in older adults with heart disease, confirming that it is beneficial and safe even in vulnerable individuals.30 Of note, we also found that the TFM increased the LF component of the HRV, implying sympathetic activation.\n\nHowever, the summed effect of the parasympathetic activity is probably more predominant, as shown by the reduced stress index. There are a few limitations in the present study. First, the majority of the volunteers were female. Generally, the RBF in females is 20% lower than in males.1 A larger sample size in a further study might be stratified into two groups to observe the gender difference. Secondly, molecular mechanisms of the TFM were not directly measured. In the other study, biochemical markers from blood samples, such as vasodilators, can be determined.31 Our analysis is confined to older adults, so generalization should be performed carefully.\n\nIn summary, by activating parasympathetic activity, the TFM may raise RBF and reduce psychological stress. As a result, this intervention may reduce stress in the senior population and enhance RBF. It is necessary to conduct additional research on a broader population.", "appendix": "Data availability\n\nOSF: Repositories for a project: Immediate effects of Thai foot massage on renal blood flow and heart rate variability in older adults. https://doi.org/10.17605/OSF.IO/YRF3J\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nThis project includes the following underlying data.\n\n- Data-1.xlsx\n\nOSF: Repositories for a project: Immediate effects of Thai foot massage on renal blood flow and heart rate variability in older adults. https://doi.org/10.17605/OSF.IO/YRF3J\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nThis project includes the following extended data.\n\n- A copy of the original trial protocol.pdf\n\n- CONSORT flow diagram.tiff\n\n- CONSORT-2010-Checklist-revised-1.pdf\n\nOSF: CONSORT-2010-Checklist-revised-1.pdf in Repositories for a project: Immediate effects of Thai foot massage on renal blood flow and heart rate variability in older adults. https://osf.io/yrf3j/\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nCONSORT flow diagram.tiff\n\n- CONSORT-2010-Checklist-revised-1.pdf\n\n\nReferences\n\nAlhummiany B, Sharma K, Buckley DL, et al.: Physiological confounders of renal blood flow measurement. MAGMA. 2023 Nov 16. Epub 20231116. PubMed Abstract \| Publisher Full Text\n\nCzarkowska-Pączek B, Mucha K, Pączek L: Age-related decline in renal blood flow could be a beneficial and compensatory mechanism. Med. Sci. Monit. 2020; 26: e918643–e918641. Publisher Full Text\n\nSalomon K: Mental Stress.Gellman MD, Turner JR, editors. Encyclopedia of Behavioral Medicine. New York, NY: Springer New York; 2013; p. 1227. Publisher Full Text\n\nD’Angelo J, Ritchie SD, Oddson B, et al.: Using heart rate variability methods for health-related outcomes in outdoor contexts: a scoping review of empirical studies. Int. J. Environ. Res. Public Health. 2023 Jan 11; 20(2). Epub 20230111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHayashi N, Someya N, Endo MY, et al.: Vasoconstriction and blood flow responses in visceral arteries to mental task in humans. Exp. Physiol. 2006 Jan; 91(1): 215–220. PubMed Abstract \| Publisher Full Text\n\nKuipers NT, Sauder CL, Carter JR, et al.: Neurovascular responses to mental stress in the supine and upright postures. J. Appl. Physiol. (1985). 2008 Apr; 104(4): 1129–1136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSripongngam T, Eungpinichpong W, Sirivongs D, et al.: Immediate Effects of Traditional Thai Massage on Psychological Stress as Indicated by Salivary Alpha-Amylase Levels in Healthy Persons. Med. Sci. Monit. Basic Res. 2015 Oct 5; 21: 216–221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSudmeier I, Bodner G, Egger I, et al.: Changes of renal blood flow during organ-associated foot reflexology measured by color Doppler sonography. Forsch. Komplementarmed. 1999; 6(3): 129–134. PubMed Abstract\n\nTudpor K: Repositories for a project: Immediate effects of Thai foot massage on renal blood flow and heart rate variability in older adults. OSF, editor. 2024. Reference Source\n\nEungpinichpong W: Therapeutic thai massage. Bang o: Chonromde Publishing House; 2008.\n\nShin Y, Ham J, Cho H: Experimental study of thermal comfort based on driver physiological signals in cooling mode under summer conditions. Appl. Sci. 2021; 11(2): 845. Publisher Full Text\n\nIsmail A, Ademola BL, Yusuf L, et al.: Renal arterial Doppler velocimetric indices among healthy subjects in north west Nigeria. J. West Afr. Coll. Surg. 2018; 8(1): 40–49. PubMed Abstract\n\nBlanco P: Volumetric blood flow measurement using Doppler ultrasound: concerns about the technique. J. Ultrasound. 2015 Jun; 18(2): 201–204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKato TS, Ono S, Kajimoto K, et al.: Early introduction of tolvaptan after cardiac surgery: a renal sparing strategy in the light of the renal resistive index measured by ultrasound. J. Cardiothorac. Surg. 2015 Nov 2; 10: 143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrunz LM, Balasubramanya R: Doppler renal assessment, protocols, and interpretation.2021.\n\nRatanakorn D, Keandaoungchan J: Volume flow rate. Pers. Med. 2012; 1(1-12): 203–206. Publisher Full Text\n\nKing BF, Torres VE, Brummer ME, et al.: Magnetic resonance measurements of renal blood flow as a marker of disease severity in autosomal-dominant polycystic kidney disease. Kidney Int. 2003 Dec; 64(6): 2214–2221. PubMed Abstract \| Publisher Full Text\n\nIwamoto K, Mizukami M, Asakawa Y, et al.: Effects of friction massage of the popliteal fossa on blood flow velocity of the popliteal vein. J. Phys. Ther. Sci. 2017 Mar; 29(3): 511–4. Epub 20170322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTudpor K, Sripongngam T, Kanpittaya J, et al.: Active ankle movements improve renal blood flow in community-dwelling elderly. J. Med. Assoc. Thail. 2019; 102(8): 51.\n\nNagaya S, Hayashi H, Fujimoto E, et al.: Passive ankle movement increases cerebral blood oxygenation in the elderly: an experimental study. BMC Nurs. 2015; 14: 14. Epub 2015/04/04. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrunz LM, Balasubramanya R: Doppler Renal Assessment, Protocols, and Interpretation. Treasure Island (FL): StatPearls; 2023. Reference Source\n\nIsmail A, Ademola BL, Yusuf L, et al.: Renal Arterial Doppler Velocimetric Indices among Healthy Subjcts in North West Nigeria. J. West Afr. Coll. Surg. 2018 Jan-Mar; 8(1): 40–49. PubMed Abstract \| Free Full Text\n\nPark J: Effects of 24-week resistance exercise training on carotid peak systolic and end diastolic flow velocity in healthy older adults. J. Phys. Ther. Sci. 2016 Oct; 28(10): 2793–2797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrudi FM, Cantisani V, Granata A, et al.: Multiparametric ultrasound in the evaluation of kidney disease in elderly. J. Ultrasound. 2020 Jun; 23(2): 115–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrikou I, Tsioufis C, Konstantinidis D, et al.: Renal resistive index in hypertensive patients. J. Clin. Hypertens. (Greenwich). 2018 Dec; 20(12): 1739–1744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTidgren B, Hjemdahl P: Renal responses to mental stress and epinephrine in humans. Am. J. Phys. 1989 Oct; 257(4 Pt 2): F682–F689. PubMed Abstract \| Publisher Full Text\n\nKim JY, Joo YS, Jhee JH, et al.: Effect of Psychosocial Distress on the Rate of Kidney Function Decline. J. Gen. Intern. Med. 2021 Oct; 36(10): 2966–2974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJing Y, Liu S, Pan C, et al.: The Effects of Foot Reflexology on Vital Signs: A Meta-Analysis of Randomized Controlled Trials. Evid. Based Complement. Alternat. Med. 2022; 2022: 4182420. Epub 20220913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen YS, Lu WA, Clemente FM, et al.: Increased parasympathetic activity by foot reflexology massage after repeated sprint test in collegiate football players: a randomised controlled trial. Sports (Basel). 2019 Nov 3; 7(11). Epub 20191103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu WA, Chen GY, Kuo CD: Foot reflexology can increase vagal modulation, decrease sympathetic modulation, and lower blood pressure in healthy subjects and patients with coronary artery disease. Altern. Ther. Health Med. 2011 Jul-Aug; 17(4): 8–14. PubMed Abstract\n\nHerrera M, Garvin JL: Recent advances in the regulation of nitric oxide in the kidney. Hypertension. 2005 Jun; 45(6): 1062–1067. PubMed Abstract \| Publisher Full Text" }	[ { "id": "308010", "date": "08 Aug 2024", "name": "Orachorn Boonla", "expertise": [ "Reviewer Expertise Cardiovascular physiology", "Oxidative stress", "Exercise physiology", "Geriatic physical therapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study demonstrated that Thai foot massage increases renal blood flow and reduces psychological stress through parasympathetic nervous activation in older adults.\nThis study provided adequate background and information to help you understand the research. The author provided adequate findings that correlate with the results and the conclusions align with the findings obtained from the study.\nHowever, the author should provide the full name of the abbreviation at the end of each figure and table such as VF, PSV, EDV, and RI. It is necessary for the author to review the results in Table 3. The results show a significant difference in HRV parameter (LF) in TFM group. The manuscript should be indexed with a few minor changes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "338484", "date": "19 Nov 2024", "name": "Enggista Hendriko Delano", "expertise": [ "Reviewer Expertise Sport Therapy", "Sport Massage", "Complementary therapy for sports 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\nReview:\nStudy this is very good , Purposeful For determine the effects of Thai foot massage on renal blood flow, psychological stress, and heart rate variability in community-dwelling older adults. Background study it's very clear Compliance between objective with method research used are already appropriate. Election sample is already determined based on criteria and quantity sample used to calculate the sample size. Does the sample take into consideration variables such as type and sex? Measuring instruments used need listed with clear along with mark validity and reliability. The intervention section needs further clarification regarding the Thai massage treatment steps, including which body parts are involved, which muscles and points are affected, and the direction of the massage. If possible, images and tables can be added to provide a clearer understanding of the treatment steps. The results section would be improved by adding a clear indication of the effectiveness based on the study's findings Clarification is needed on why massage on the soles of the feet can provide effective therapy. This should be explained and supplemented with the relevant physiological mechanisms of the body.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/13-838
https://f1000research.com/articles/10-830/v1	19 Aug 21	{ "type": "Software Tool Article", "title": "UKB.COVID19: an R package for UK Biobank COVID-19 data processing and analysis", "authors": [ "Longfei Wang", "Victoria E Jackson", "Liam G Fearnley", "Melanie Bahlo", "Victoria E Jackson", "Liam G Fearnley", "Melanie Bahlo" ], "abstract": "COVID-19 caused by SARS-CoV-2 has resulted in a global pandemic with a rapidly developing global health and economic crisis. Variations in the disease have been observed and have been associated with the genomic sequence of either the human host or the pathogen. Worldwide scientists scrambled initially to recruit patient cohorts to try and identify risk factors. A resource that presented itself early on was the UK Biobank (UKBB), which is investigating the respective contributions of genetic predisposition and environmental exposure to the development of disease. To enable COVID-19 studies, UKBB is now receiving COVID-19 test data for their participants every two weeks. In addition, UKBB is delivering more frequent updates of death and hospital inpatient data (including critical care admissions) on the UKBB Data Portal. This frequently changing dataset requires a tool that can rapidly process and analyse up-to-date data. We developed an R package specifically for the UKBB COVID-19 data, which summarises COVID-19 test results, performs association tests between COVID-19 susceptibility/severity and potential risk factors such as age, sex, blood type, comorbidities and generates input files for genome-wide association studies (GWAS). By applying the R package to data released in April 2021, we found that age, body mass index, socioeconomic status and smoking are positively associated with COVID-19 susceptibility, severity, and mortality. Males are at a higher risk of COVID-19 infection than females. People staying in aged care homes have a higher chance of being exposed to SARS-CoV-2. By performing GWAS, we replicated the 3p21.31 genetic finding for COVID-19 susceptibility and severity. The ability to iteratively perform such analyses is highly relevant since the UKBB data is updated frequently. As a caveat, users must arrange their own access to the UKBB data to use the R package.", "keywords": [ "R package", "UK Biobank", "COVID-19", "GWAS", "risk factors" ], "content": "Introduction\n\nThe ongoing global pandemic of coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), has resulted in a rapidly developing global health and economic crisis. Most people with COVID-19 never develop symptoms or suffer mild symptoms. However, about 5% of cases are critical (defined as respiratory failure, septic shock, and/or multiorgan dysfunction or failure) (Wu and McGoogan 2020), possibly leading to lethal lung damage and even death. These and other clinical observations led to the hypothesis that genetic factors in either or both the host and the pathogen could be responsible, at least in part, for this variation. Worldwide scientists scrambled initially to recruit patient cohorts to try and identify genetic risk factors.\n\nUK Biobank (UKBB) (RRID: SCR_012815) is a long-term biobank study that recruited 500,000 volunteers aged between 40–69 years in 2006–2010 from across the UK. UKBB’s large-scale database is a global research resource accessible to approved researchers who are undertaking health-related research. All participants provided detailed information about their lifestyle, physical measures and had blood, urine and saliva samples collected. The samples of all participants have undergone SNP array typing and are now also undergoing whole-exome and whole-genome sequencing. UKBB has become a major contributor to the advancement of modern medicine and treatment, enabling a better understanding of a wide range of serious and life-threatening diseases.\n\nResearchers can apply for access to the data and worldwide hundreds of researchers are using the UKBB data to carry out research on many different diseases. The UKBB has facilitated first-time analyses on traits such as brain imaging phenotypes (Elliott et al., 2018).\n\nThe UK has been badly affected by COVID-19. As of 20 May 2021, there have been over 127,000 reported deaths in the UK, with an estimated 4.5 million infections. Worldwide there have now been more than 3 million reported deaths due to COVID-19, with continually increasing rates of infections in India and South America. The UKBB was an early, available population genetic resource that could be harnessed to better understand COVID-19 risk factors, and with its continuing evolution continues to serve as a powerful cohort to permit such studies.\n\nUKBB has taken swift strides to help tackle the global pandemic by undertaking four major initiatives: serology study, COVID-19 repeat imaging study, coronavirus self-test antibody study and health data linkage. UKBB has been receiving COVID-19 test data for previous UKBB participants in England and has linked the test result data with health data. The test results data are being updated every two weeks. In addition, UKBB is making more frequent updates of death and hospital inpatient data (including critical care admissions) on the Data Portal. This rapidly changing dataset requires a tool that can process the up-to-date data as frequently as the data updates, in a standardised, reproducible, and somewhat automated manner to permit rapid re-analysis of the data and to also enable other researchers to use such a tool as a basis for their analyses.\n\nTherefore, we developed an R package (version 4.0.5) UKB.COVID-19 which summarises COVID-19 test results, combines test results data with hospitalisation data and death register data, performs association tests between COVID-19 susceptibility/severity and potential risk factors (age, sex, blood type, socioeconomic status, comorbidities etc.) and generates input files for genome-wide association studies (GWAS). Ethics approval was granted through WEHI project 17/09LR by the WEHI’s Human Research Ethics Committee (HREC).\n\n\nMethods\n\nUKB.COVID19 was built in R (version 4.0.5) and currently depends on the following R packages: questionr, data.table, tidyverse, magrittr, here, and dplyr. COVID-19 related data files from UKBB can be directly imported in the R package without any pre-processing.\n\nUKB.COVID19 is distributed as part of the CRAN R package repository and is compatible with Mac OS X, Windows, and major Linux operating systems. UKB.COVID19 is maintained at GitHub (https://github.com/bahlolab/UKB.COVID19). The archived source code can be found in http://doi.org/10.5281/zenodo.5174381 (Wang et al., 2021). All analyses are performed using R (version 4.0.5). All functions and descriptions are listed in Table 1.\n\nCOVID-19 test results data are being provided to the UKBB by Public Health England (PHE), Public Health Scotland (PHS) and SAIL Databank for English, Scottish and Welsh data respectively. The data have been updated approximately once every two weeks since 16 March 2020. Most samples tested for the COVID-19 disease-causing virus, SARS-CoV-2, are from combined nose/throat swabs. In intensive care settings, lower respiratory tract samples may also have been taken and analysed. The data consists of the encoded participant ID, date the specimen was taken, specimen type (e.g. nasal, nose and throat, sputum), the laboratory that processed the sample, whether the sample was reported as positive or negative for SARS-CoV-2, the requesting organisation description, as well as other variables. The test result data used in the analyses of this report are up to 6 April 2021.\n\nThe death register data includes the date of death, the primary and contributory causes of death, coded using the ICD-10 system. The death register data have been updated every one or two months. The death register data used in the analyses of this report are up to 23 March 2021.\n\nThe hospital inpatient data consist of seven tables: 1) HESIN: the overall master table, providing information on admissions and discharges, the type of admission and other information related to the inpatient record as a whole. 2) HESIN_DIAG: diagnosis codes (ICD-9 or ICD-10) relating to inpatient records, including primary diagnoses and secondary diagnoses. The primary diagnosis is the main condition treated or investigated during the relevant episode. A secondary diagnosis is a clinically relevant contributory factor or issue that impacts the primary diagnosis (including chronic conditions). 3) HESIN_OPER: operations and procedures codes (OPCS-3 or OPCS-4) relating to inpatient episodes. 4) HESIN_CRITICAL: a child table of HESIN containing further information about those hospital episodes that required treatment in a critical care unit. 5) HESIN_PSYCH: a sibling table to HESIN containing fields relating to administrative aspects of psychiatric admissions. 6) HESIN_MATERNITY: a sibling table to HESIN containing fields relating specifically to maternity admissions. 7) HESIN_DELIVERY: Information regarding a child born as a result of a HESIN_MATERNITY record, where applicable. In this study, we use the HESIN, the HESIN_DIAG, the HESIN_OPER, and the HESIN_CRITICAL tables. The hospital inpatient data used in the analyses of this report are up to 5 February 2021.\n\nThe makePhenotypes function defines multiple COVID-19 traits, related to susceptibility, severity and mortality, which may be used for association testing and GWAS (Table 2).\n\nThe COVID-19 related phenotypes output from the makePhenotypes function in the UKB.COVID19 R package.\n\nFor susceptibility analysis, we generated a proxy variable, which includes all participants who have been tested for COVID-19 and define those who received at least one positive result as cases. By 6 April 2021, 77,222 individuals in the UKBB had received COVID-19 tests and 16,562 had tested positive for COVID-19 on at least one occasion. The pheno.type = “susceptibility” option summarises the COVID-19 test results data and generates a susceptibility phenotype for association tests and GWAS.\n\nBased on the World Health Organization (WHO) ordinal scale for clinical improvement, we classify severity into three levels. These levels are defined as 1) hospitalisation: individuals admitted to hospital with their primary diagnosis recorded as COVID-19. 2) critical care: individuals that required treatment in a critical care unit, such as non-invasive ventilation and continuous positive airway pressure, and with their primary diagnosis recorded as COVID-19. 3) advanced critical care: individuals required advanced treatment in a critical care unit, such as invasive ventilation and temporary tracheostomy, and with their primary diagnosis recorded as COVID-19. The critical care information was summarised from the HESIN_CRITICAL table and the HESIN_OPER table. We compare the test results data and the hospital inpatient data and correct any inconsistency between the two tables. As an example of data inconsistency, up to 5 February 2021, 130 individuals were admitted to the hospital due to COVID-19 but are not recorded in the test result data, while 33 individuals were admitted to the hospital due to COVID-19 but received negative COVID-19 test results. This inconsistency is resolved by retaining all 163 individuals and setting their COVID-19 test results as positive. The pheno.type = “severity” option combines COVID-19 test results data and hospital inpatient data and generates three phenotypes for each severity level.\n\nFor mortality, we include all individuals who received at least one positive test result and define those whose primary cause of death is recorded as being due to COVID-19 as cases. We also compare the test results data and the death register data and correct any inconsistencies. As an example, up to 23 March 2021, 205 individuals died from COVID-19 as reported by the death register data but are not recorded as having positive COVID-19 tests in the test result data while 39 individuals died from COVID-19 but received negative COVID-19 test results. The inconsistency is resolved by retaining all 244 individuals and setting their test results as positive. Therefore, in total 1,042 UKBB participants had died from COVID-19 by 23 March 2021. The pheno.type = “mortality” option combines the COVID-19 test results data and death register data and generates a mortality phenotype.\n\nThe makePhenotypes function returns results in data.frame format and outputs files in text format for the downstream association tests and genome-wide association tests using PLINK (RRID:SCR_001757) (Purcell et al., 2007) and SAIGE (Scalable and Accurate Implementation of GEneralized mixed model) (Zhou et al., 2018).\n\nThe risk.factor function generates formatted variables for several non-genetic risk factors from the linked health data provided by UKBB. These variables are all established risk factors for SARS-CoV-2 exposure, and/or COVID-19 severity (Pijls et al., 2021; Wolff et al., 2021; Booth et al., 2021). The currently selected risk factors are listed in Table 3. The multi-category variables are converted into multiple dummy variables. For the blood type group factor, three dummy variables encoding the blood types A, AB, and O, are added to the data to compare with blood type B (baseline). For the ethnic background factor, Black, Asian, Mixed, and other ethnic backgrounds (BAME) are added to the data to permit comparison to white Europeans (baseline).\n\nSimple associations between COVID-19 phenotypes and these common risk factors may be examined using the log_cov function, which performs a logistic regression model and formats the results for quick interpretation.\n\nThe comorbidity.summary function summarises disease history records of each individual from the hospital inpatient diagnosis data. To meet different research aims the function allows restriction to a period and filtering of annotations by only primary diagnoses or all diagnoses (using the \"Date.start\", \"Date.end\" and \"primary\" arguments, respectively). For illustration, if we are interested in the co-occurrences of COVID-19, we can set the episode start date as 16 March 2020 (“Date.start = 16/03/2020”), when the first COVID-19 test result was recorded and choose to use all diagnoses (“primary = FALSE”). If we are interested in individuals with reported comorbidities that are at a higher risk to SARS-CoV-2, we can choose an episode start time before the COVID-19 outbreak in the UK, for example, “Date.end = 01/01/2020” and only focus on the primary diagnoses (“primary = TRUE”). Comorbidity categories are generated using the block categories in the ICD10 code. For instance, the first category is “A00-A09”, representing intestinal infectious diseases. The comorbidity categories include ICD10 chapters 1–14 and 17 and exclude several categories such as pregnancy, pregnancy delivery, consequences of external causes etc. The R function generates a text file including all comorbidities, which can be used in the comorbidity association tests.\n\nThe comorbidity.asso function performs association tests using logistic regression models for each comorbidity and adjusts the tested phenotype with covariates, which can be set using the argument “cov.name”. By default, the covariates include sex, age, and BMI. Different ethnic backgrounds can be chosen for the test by setting the argument “population”. By default, all populations are included. It outputs a table comprised of odds ratios (ORs), confidence intervals (CIs) of ORs, and p-values for all the comorbidity categories.\n\nThe UKB.COVID19 package provides several functions, to facilitate GWAS, or other genetic analyses using the UKBB data. We provide two functions sampleQC and variantQC, to allow easy cleaning of the genetic data, using quality control (QC) metrics, supplied by UKBB (Bycroft et al., 2018). A third function, makeGWASFiles, outputs phenotype files, which may be used as input for the GWAS software packages PLINK (Purcell et al., 2007) and SAIGE (Zhou et al., 2018).\n\nThe sampleQC function outputs a csv file summarising sample-level QC metrics, as well as producing lists of IDs for inclusion and/or exclusion in downstream analyses. The function identifies individuals to be excluded from genetic analyses based on: 1) being excluded by UKBB, before imputation due to high heterozygosity or missingness (>5%), 2) sex mismatches between genetically predicted and recorded sex, 3) an apparent excess number of relatives in the UKBB cohort (≥ 10 relatives), 4) putative sex chromosome aneuploidy, 5) withdrawn consent. The user has the option of further restricting to individuals of “White British” ancestry (determined using genetic principal components), by using the ancestry argument. Finally, the user can specify whether they require inclusion/exclusion sample lists to be formatted for PLINK or SAIGE.\n\nThe variantQC function identifies variants to be included in downstream analyses, based on minor allele frequency (MAF) and imputation quality (INFO score), with thresholds specified by the user (defaults to MAF ≥0.001 and INFO ≥0.5). The function outputs list of variants passing these thresholds are in two formats, given the two types of SNP IDs available in the UKBB imputed genetic data release: 1) snpIncludeSNPIDs_minMaf0.001_minInfo0.5.txt contains the unique SNP identifiers; 2) snpIncludeRSIDs_minMaf0.001_minInfo0.5.txt contains the rsid or the reference panel marker ID (note these IDs are not guaranteed to be unique). The function also outputs a file containing IDs of the subset of SNPs, used by UKBB for calculating ancestry principal components (Bycroft et al., 2018). This subset of SNPs is suitable for analyses where a pruned set of independent SNPs are preferred, for example for calculation of a genetic relatedness matrix (GRM).\n\nThe makeGWASFiles function generates a phenotype file, suitable to be used in association analyses by either SAIGE or PLINK (Purcell et al., 2007) (File format specified by user). The function utilises the phenotypes data frame generated by the makePhenotypes function, with the user able to specify specific phenotypes. The output phenotype file also contains the first 20 ancestry principal components, and genotyping array, as these are likely to be required as covariates in any genetic analyses. The user can also specify additional covariates (e.g. those generated by the risk.factor function), to be outputted to the phenotype file. Finally, the user can choose to output phenotypes, only for the individuals passing all QC (using the output file from sampleQC function), or for all individuals.\n\nWe performed QC for the genotype data from UKBB using the sampleQC function, with the ancestry = “WhiteBritish” option, and the variantQC function, with thresholds MAF = 0.01 and INFO = 0.8. Phenotype files for SAIGE were generated using the makeGWASFiles function, containing all variables generated by the risk.factor function.\n\nUsing the output files from the sampleQC and variantQC functions, we filtered the directly genotyped data using PLINK (Purcell et al., 2007), and the imputed data using QCTool version 2. We then performed GWAS of all COVID-19 phenotypes using SAIGE (Zhou et al., 2018). Firstly, the null model was fitted for each phenotype with 20 ancestry procedure codes (PCs), genotypic array, and associated non-genetic risk factors as covariates, and we used the pruned subset SNPs to construct the GRM. Subsequently, genome-wide association testing was undertaken, using the filtered imputed data.\n\n\nResults\n\nWe applied the R package UKB.COVID19 to the data released in April 2021. The last records in the COVID-19 test results data, the death register data and the hospital inpatient data were recorded on 6 April 2021, 23 March 2021, and 5 February 2021, respectively. By default, the dates for susceptibility, severity and mortality studies were chosen as 6 April 2021, 5 February 2021, and 23 March 2021, accordingly.\n\nBy 6 April 2021, 77,222 UKBB participants had tested for COVID-19. Among these individuals, 16,562 received at least one positive test result and 60,660 received all negative results. First, we tested the associations between a positive test result (as a proxy for COVID-19 susceptibility), and age, sex, and BMI using multivariable logistic regression. The results (Table 4) show increased odds of a positive result in individuals of male sex (OR = 1.08, 95% CI = [1.04,1.11], p-value = 0.00007), with higher BMI (OR = 1.026, 95% CI = [1.0229,1.03], p-value <10−5) and with younger ages (OR = 0.939, 95% CI = [0.937,0.941], p-value <10−5). A possible reason for this result is that the older participants are less active and thus had less chance of being exposed to SARS-CoV-2.\n\nCases are defined as participants who received at least one COVID-19 positive test result. Controls are those who received only negative results. We tested sex, age and body mass index (BMI) in a multivariable model first and then tested each other factor individually by adjusting sex, age and BMI. SES stands for socioeconomic status. Odds ratio (OR) and p-values (P) are provided.\n\n≈0 means <10−5.\n\nSecond, we tested each potential risk factor individually with adjustment of age, sex, and BMI. Several publications have already reported that blood type groups are associated with COVID-19 susceptibility (Zhao et al., 2020; Zietz, Zucker, and Tatonetti 2020), including genetic associations with the ABO blood group locus at 9q34.2 (The Severe Covid-19 GWAS Group “Genomewide Association Study of Severe Covid-19 with Respiratory Failure” 2020). People with blood type A have been consistently reported as being at a higher risk to SARS-CoV-2 and people with blood type O at lower risk (Zhao et al., 2020). Consistent with these results we find that compared with type B, individuals with blood type O are less susceptible to SARS-CoV-2 (OR =0.91, 95% CI = [0.86,0.97], p-value = 0.005) but we were unable to replicate the type A findings (p-value = 0.7).\n\nCompared with white individuals, those who self-identified as Black (OR =1.38, 95% CI = [1.24,1.55], p-value <10−5), Asian (OR =1.88, 95% CI = [1.71,2.07], p-value <10−5) and other ethnic backgrounds (OR =1.33, 95% CI = [1.14,1.55], p-value =0.0004) have higher odds of testing positive for COVID-19. Individuals with a lower socioeconomic status (SES) are also at a higher risk of COVID-19 (OR = 1.041, 95% CI = [1.036,1.047], p-value <10−5). Smoking also contributes to COVID-19 susceptibility (OR =1.003, 95% CI = [1.002,1.004], p-value <10−5). People who are staying at an aged care home are at a significantly higher risk of COVID-19 (OR = 2.13, 95% CI = [1.87,2.43], p-value <10−5), which is in line with the aged care home outbreaks in the UK.\n\nWe only apply GWAS to the white British participants in the UKBB. Therefore, we performed non-genetic risk factor association tests again for self-reported “white” participants only. It shows that age, sex, BMI, SES, smoking, and if in an aged care home are associated with COVID-19 susceptibility in white British. Incorporation of the two array effects and the first 20 PCs, these risk factors are used to adjust susceptibility in the GWAS. The genome-wide significant COVID-19 susceptibility locus identified in our GWAS is 3p21.31 (Figure 1 and Table 5). The most statistically significant SNP is rs2771616 within the glycine transporter gene SLC6A20 (3p21.31, p-value = 3.36 × 10−9), followed by SNPs rs73062389 (3p21.31; SLC6A20; p-value =5.16 × 10−9) and rs73062394 (3p21.31; SLC6A20; p-value = 6.68 × 10−9) in strong linkage disequilibrium (LD) (r2 = 1 and r2 = 1) (Table 6). SLC6A20 encodes an amino acid transporter that interacts with ACE2, the main receptor that SARS-CoV-2 uses to gain entry into host cells (Elhabyan et al., 2020; Hoffmann et al., 2020). This locus has also been previously identified by other studies (The Severe Covid-19 GWAS Group “Genomewide Association Study of Severe Covid-19 with Respiratory Failure”, 2020), several meta-analyses of which have also made use of the UKBB COVID-19 data (Host Genetics Initiative, 2021). All genome wide significant GWAS hits with gene annotations are available in Table 6.\n\nSample size is 61,823. In the Manhattan plot, each point denotes a SNP located on a particular chromosome (x-axis). The significance level is presented in the y-axis. The red line indicates the threshold for genome-wide significance 5 × 10−8 while the blue line indicates the threshold for suggestive genome-wide significance 1 × 10−5. The light green dots are the genes of interest, which have been reported in other publications (Pairo-Castineira et al., 2021; “Genomewide Association Study of Severe Covid-19 with Respiratory Failure”, 2020), including SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, HLA-G, CCHCR1, NOTCH4, ABO, OAS1, OAS2, OAS3, APOE, DPP9, TYK2, IFNAR2, TMPRSS2, ACE2, and TLR7. The susceptibility phenotype is adjusted by age, sex, body mass index, socioeconomic status, smoking, if in an aged care home, array, and PC1–20. The genome-wide significant COVID-19 susceptibility locus identified is 3p21.31. The most statistically significant SNP is rs2771616 within the glycine transporter gene SLC6A20 (3p21.31, p-value =3.36 × 10−9), followed by SNPs rs73062389 (3p21.31; SLC6A20; p-value = 5.16 × 10−9) and rs73062394 (3p21.31; SLC6A20; p-value = 6.68 × 10−9) in strong linkage disequilibrium (LD) (r2 = 1 and r2 = 1).\n\nThe most genome-wide significant hits of COVID-19 susceptibility, hospitalisation and critical care genome-wide association studies.\n\nThe genome-wide significant hits of COVID-19 susceptibility, hospitalisation and critical care genome-wide association studies.\n\nBy 5 February 2021, 15,666 UKBB participants received positive COVID-19 test results. 2,104 individuals had been admitted to the hospital due to COVID-19, 1,129 of these individuals received critical care treatments and 1,010 received advanced critical care treatments. The risk factor association test results are presented in Tables 7 and 8 for all populations and self-reported white individuals, respectively. Compared to white individuals, Black, Asian, and other minority ethnic groups are at a higher risk of severe COVID-19. Age, sex, BMI, SES, and smoking are also positively associated with COVID-19 severity.\n\nCases of hospitalisation include participants who were admitted to hospital and whose primary diagnosis was COVID-19, received critical care treatments, or died from COVID-19. Controls are the rest of the participants who received positive test results. Cases of critical care phenotype include those who received critical care treatments due to COVID-19 or died from COVID-19. Cases of advanced critical care are defined as participants who received advanced critical care treatments or died from COVID-19. We tested sex, age and body mass index (BMI) in a multivariable model first and then tested each other factor individually by adjusting sex, age and BMI. SES stands for socioeconomic status. Odds ratio (OR) and p-values (P) are provided.\n\n≈0 means <10−5.\n\nCases of hospitalisation include participants who were admitted to hospital and whose primary diagnosis was COVID-19, received critical care treatments, or died from COVID-19. Controls are the rest of the participants who received positive test results. Cases of critical care phenotype include those who received critical care treatments due to COVID-19 or died from COVID-19. Cases of advanced critical care are defined as participants who received advanced critical care treatments or died from COVID-19. We tested sex, age and body mass index (BMI) in a multivariable model first and then tested each other factor individually by adjusting sex, age and BMI. SES stands for socioeconomic status. Odds ratio (OR) and p-values (P) are provided.\n\n≈0 means <10−5.\n\nThe results from the GWAS are shown in the quantile-quantile (Q-Q) plots and Manhattan plots in Figures 2–4. The tested phenotypes are adjusted by age, sex, BMI, SES, smoking, if in an aged care home, array, and PC1–20. The results show that the locus at 3p21.31 is genome-wide significantly associated with COVID-19 hospitalisation and critical care (Tables 5 and 6). Specifically, the most significant SNP for both COVID-19 hospitalisation and critical care GWASs is located in the gene LZTFL1 (rs35044562 in locus 3p21.31; p-value = 1.55 × 10−10 and p-value = 2.23 × 10−9, respectively). According to the Genotype-Tissue Expression (GTEx) project, LZTFL1 is widely expressed throughout the body and encodes a protein involved in protein trafficking to primary cilia, which are microtubule-based subcellular organelles acting as antennas for extracellular signals. In T lymphocytes, LZTFL1 participates in the immunologic synapse with antigen-presenting cells, such as dendritic cells (these cells prime T-lymphocyte responses) (Kaser 2020; Seo et al., 2011; Jiang et al., 2016).\n\nSample size is 11,974. In the Manhattan plot, each point denotes a SNP located on a particular chromosome (x-axis). The significance level is presented in the y-axis. The red line indicates the threshold for genome-wide significance 5 × 10−8 while the blue line indicates the threshold for suggestive genome-wide significance 1 × 10−5. The light green dots are the genes of interest, including SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, HLA-G, CCHCR1, NOTCH4, ABO, OAS1, OAS2, OAS3, APOE, DPP9, TYK2, IFNAR2, TMPRSS2, ACE2, and TLR7. The hospitalisation phenotype is adjusted by age, sex, body mass index, socioeconomic status, smoking, if in an aged care home, array, and PC1–20. The result shows that the locus at 3p21.31 is genome-wide significantly associated with COVID-19 hospitalisation. The most significant SNP for both COVID-19 hospitalisation GWAS is located in the gene LZTFL1 (rs35044562 in locus 3p21.31; p-value = 1.55 × 10−10).\n\nSample size is 11,974. In the Manhattan plot, each point denotes a SNP located on a particular chromosome (x-axis). The significance level is presented in the y-axis. The red line indicates the threshold for genome-wide significance 5 × 10−8 while the blue line indicates the threshold for suggestive genome-wide significance 1 × 10−5. The light green dots are the genes of interest, including SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, HLA-G, CCHCR1, NOTCH4, ABO, OAS1, OAS2, OAS3, APOE, DPP9, TYK2, IFNAR2, TMPRSS2, ACE2, and TLR7. The critical care phenotype is adjusted by age, sex, body mass index, socioeconomic status, smoking, if in an aged care home, array, and PC1–20. The result shows that the locus at 3p21.31 is genome-wide significantly associated with COVID-19 critical care. The most significant SNP for both COVID-19 critical care GWAS is located in the gene LZTFL1 (rs35044562 in locus 3p21.31; p-value = 2.23 × 10−9).\n\nSample size is 11,974. In the Manhattan plot, each point denotes a SNP located on a particular chromosome (x-axis). The significance level is presented in the y-axis. The red line indicates the threshold for genome-wide significance 5 × 10−8 while the blue line indicates the threshold for suggestive genome-wide significance 1 × 10−5. The light green dots are the genes of interest, including SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, HLA-G, CCHCR1, NOTCH4, ABO, OAS1, OAS2, OAS3, APOE, DPP9, TYK2, IFNAR2, TMPRSS2, ACE2, and TLR7. The advanced critical care phenotype is adjusted by age, sex, body mass index, socioeconomic status, smoking, if in an aged care home, array, and PC1–20. No genome-wide significant signals were found.\n\nBy 23 March 2021, 16,465 UKBB participants received positive COVID-19 test results. Among these, 1,042 individuals died from COVID-19. We performed the same association tests for COVID-19 mortality as for susceptibility and severity. The results (Table 9) show that males have a much higher chance of dying from COVID-19 than females (OR = 1.89, 95% CI = [1.63,2.20], p-value <10−5), consistent with previously published results from independent cohorts (Peckham et al., 2020). The black ethnic group is at a much higher mortality risk from SARS-CoV-2 compared to white individuals (OR = 2.04, 95% CI = [1.38,2.94], p-value = 0.0002). Age, BMI, SES, and smoking are positively associated with COVID-19 mortality. People living in aged care homes are at a much higher risk of dying from COVID-19. For self-reported white individuals, age, sex, BMI, SES, smoking, and being in an aged care home are positively associated with COVID-19 mortality. Therefore, all these covariates were used to adjust the mortality phenotype for GWAS. However, no genome-wide significant signal was detected for this GWAS (Figure 5).\n\nCases of mortality include participants whose primary death cause is COVID-19. Controls are the rest of the participants who received positive test results. We tested sex, age and body mass index (BMI) in a multivariable model first and then tested each other factor individually by adjusting sex, age and BMI. SES stands for socioeconomic status. Odds ratio (OR) and p-values (P) are provided.\n\n≈0 means <10−5.\n\nSample size is 12,790. In the Manhattan plot, each point denotes a SNP located on a particular chromosome (x-axis). The significance level is presented in the y-axis. The red line indicates the threshold for genome-wide significance 5 × 10−8 while the blue line indicates the threshold for suggestive genome-wide significance 1 × 10−5. The light green dots are the genes of interest, including SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, HLA-G, CCHCR1, NOTCH4, ABO, OAS1, OAS2, OAS3, APOE, DPP9, TYK2, IFNAR2, TMPRSS2, ACE2, and TLR7. The mortality phenotype is adjusted by age, sex, body mass index, socioeconomic status, smoking, if in an aged care home, array, and PC1–20. No genome-wide significant signals were found.\n\nWe were interested in the co-occurrence of COVID-19 and comorbidities in individuals who had suffered from severe COVID-19. Therefore, we divided the hospital inpatient diagnosis records into before and after the COVID-19 pandemic using the date 16 March 2020, when COVID-19 testing commenced in the UK. We performed association testing for each comorbidity using logistic regression models and adjusted COVID-19 severity (if the patient received critical care treatments) by sex, age, BMI, SES, smoking and aged care status. Tables 10 and 11 list the top ten associated diseases with severe COVID-19 before and after 16 March 2020. respectively. From Table 11, we found that the common co-occurrence associated with COVID-19 are pneumonia, respiratory diseases, renal failure, metabolic disorders, hypertensive diseases, heart disease and other bacterial diseases. People who have ever had mental disorders, influenza and pneumonia, renal failure, respiratory diseases, bacterial, viral, or other infections, malignant neoplasms of lymphoid, haematopoietic and related tissue, or other blood diseases, tend to have severe symptoms after being infected by SARS-CoV-2.\n\nWe divided the hospital inpatient diagnosis records into before and after the COVID-19 pandemic using the date 16 March 2020, when COVID-19 testing commenced. We performed association testing for each comorbidity using logistic regression models and adjusted COVID-19 severity (if the patient received critical care treatments) by sex, age, body mass index, socioeconomic status, smoking and aged care status. To show the comorbidities in individuals who had suffered from severe COVID-19, we ranked the p-values before 16 March 2020 and listed the top 10 comorbidities.\n\nWe divided the hospital inpatient diagnosis records into before and after the COVID-19 pandemic using the date 16 March 2020, when COVID-19 testing commenced. We performed association testing for each comorbidity using logistic regression models and adjusted COVID-19 severity (if the patient received critical care treatments) by sex, age, body mass index, socioeconomic status, smoking and aged care status. To show the top 10 co-occurrence of COVID-19, we ranked the p-values after 16 March 2020 and listed the top 10 comorbidities.\n\nSeveral publications have reported that the APOE e4 genotype is associated with COVID-19 susceptibility and severity (Numbers and Brodaty 2021; Kuo et al., 2020a, 2020b). APOE e4 is a known risk factor for dementia, which has been replicated many times (Liu et al., 2013; Safieh, Korczyn, and Michaelson 2019; Emrani et al., 2020). One explanation for people with APOE e4 being at higher risk of COVID-19 could be due to a higher risk of exposure, as these individuals are more likely to reside in care homes, which have suffered from high rates of infections. This is particularly likely to be the case in UKBB, where 47% of participants are older than 70 years old. To test this hypothesis, we performed GWAS tests with and without aged care status. The APOE e4 signal was genome-wide significant without aged care status but was gone after aged care status adjustment (Figure 6), suggesting that this finding is not robust and may be due to ascertainment bias.\n\na. COVID-19 susceptibility GWAS without care home status covariate adjustment. The model we used is: susceptibility ~ age + sex + BMI + PC1-20 + array + SNP. b. COVID-19 susceptibility GWAS with care home status covariate adjustment. The model we used is: susceptibility ~ age + sex + BMI + PC1-20 + array + inAgedCare + SNP. The APOE e4 signal was genome-wide significant without aged care status but was gone after aged care status adjustment, suggesting that this finding is not robust and may be due to ascertainment bias.\n\n\nUse cases\n\nTo demonstrate the functionality and utility of UKB.COVID19, we present a basic tutorial for using UKB.COVID19. Due to the restriction of using UKBB data, we illustrate the use cases using simulated data. The SAIGE GWAS script example can be found in Github: https://github.com/bahlolab/UKB.COVID19/tree/main/GWAS.\n\nGenerating a covariate file. The risk.factor function in UKB.COVID19 can be used to generate a covariate file with established risk factors and risk factors of interest by specifying the field code in UKBB main data.\n\nlibrary (UKB.COVID19)\n\ncovar <- risk.factor (ukb.data=covid_example(\"sim_ukb.tab.gz\"),\n\nABO.data=covid_example(\"sim_covid19_misc.txt.gz\"),\n\nhesin.file=covid_example(\"sim_hesin.txt.gz\"),\n\nres.eng=covid_example(\"sim_result_england.txt.gz\"),\n\nout.file=paste0(covid_example(\"results\"),\"/covariate\"))\n\nhead (covar)\n\n#> ID sex age bmi ethnic other.ppl black asian mixed white SES smoke blood_group O AB B A inAgedCare\n\n#> 1 1 1 74 39.0947 1001 0 0 0 0 1 5.43719 0.000 AO 0 0 0 1 0\n\n#> 2 2 1 58 25.3177 1001 0 0 0 0 1 2.10787 0.000 AO 0 0 0 1 0\n\n#> 3 3 0 51 32.2349 1002 0 0 0 0 1 7.36321 25.625 AO 0 0 0 1 0\n\n#> 4 4 0 56 21.7955 1001 0 0 0 0 1 5.62047 0.000 AO 0 0 0 1 0\n\n#> 6 6 1 67 25.9823 1001 0 0 0 0 1 3.90245 0.000 OO 1 0 0 0 0\n\nGenerating COVID-19 susceptibility phenotype file with risk factors. In the output file, columns “pos.neg” and “pos.ppl” are the susceptibility phenotypes, which denote 1) UKBB participants with COVID-19 positive versus negative results 2) and participants with positive results versus all the other participants.\n\nphe <- makePhenotypes (ukb.data=covid_example(\"sim_ukb.tab.gz\"),\n\nres.eng=covid_example(\"sim_result_england.txt.gz\"),\n\ndeath.file=covid_example(\"sim_death.txt.gz\"),\n\ndeath.cause.file=covid_example(\"sim_death_cause.txt.gz\"),\n\nhesin.file=covid_example(\"sim_hesin.txt.gz\"),\n\nhesin_diag.file=covid_example(\"sim_hesin_diag.txt.gz\"),\n\nhesin_oper.file=covid_example(\"sim_hesin_oper.txt.gz\"),\n\nhesin_critical.file=covid_example(\"sim_hesin_critical.txt.gz\"),\n\ncode.file=covid_example(\"coding240.txt.gz\"),\n\npheno.type = \"susceptibility\",\n\nout.name=paste0(covid_example(\"results\"),\"/phenotype\"))\n\n#> [1] \"965 participants got tested until 2021-04-05.\"\n\n#> [1] \"218 participants got positive test results until 2021-04-05.\"\n\n#> [1] \"There are 21 deaths with COVID-19. 20 of them primary death cause is COVID-19.\"\n\n#> [1] \"50 patients admitted to hospital were diagnosed as COVID-19 until 2021-04-05.\"\n\n#> [1] \"32 patients' primary diagnosis is COVID-19.\"\n\n#> [1] \"1 patients in hospitalisation with COVID-19 diagnosis but show negative in the result file. Modified their test results.\"\n\n#> [1] \"There are 219 COVID-19 patients identified. 32 individuals are admitted to hospital. 3 had been in ICU. 1 had been in advanced ICU.\"\n\n#> [1] \"Outputting file: ~/UKB.COVID19/extdata/results/phenotype.txt\"\n\nhead (phe)\n\n#> ID pos.neg pos.ppl\n\n#> 1 1 1 1\n\n#> 2 2 0 0\n\n#> 3 3 0 0\n\n#> 4 4 0 0\n\n#> 5 5 0 0\n\n#> 6 6 0 0\n\nPerforming association tests. The log_cov function performs association tests using logistic regressions. This is an example of association tests between COVID-19 susceptibility and three risk factors: sex, age and BMI.\n\nlog_cov(pheno=phe, covariates=covar, phe.name=\"pos.neg\", cov.name=c(\"sex\", \"age\", \"bmi\"))\n\n#> Estimate OR 2.5 % 97.5 % p\n\n#> (Intercept) -0.16475743 0.8480994 0.1954585 3.6381032 0.824991899\n\n#> sex1 0.04207813 1.0429760 0.7644672 1.4215535 0.790121307\n\n#> age -0.03080456 0.9696651 0.9519878 0.9876397 0.001009957\n\n#> bmi 0.03625193 1.0369170 1.0076088 1.0667564 0.012568486\n\nGenerating a comorbidity summary file. The comorbidity.summary function scans all the hospitalisation records with a given time period and generates a text file. The following example is to generate a comorbidity summary file that includes all the primary and secondary diagnoses in the hospital inpatient data after 16 March 2020.\n\ncomorb <- comorbidity.summary (ukb.data=covid_example(\"sim_ukb.tab.gz\"),\n\nhesin.file=covid_example(\"sim_hesin.txt.gz\"),\n\nhesin_diag.file=covid_example(\"sim_hesin_diag.txt.gz\"),\n\nICD10.file=covid_example(\"ICD10.coding19.txt.gz\"),\n\nprimary = FALSE,\n\nDate.start = \"16/03/2020\",\n\noutfile=paste0(covid_example(\"results\"),\"/comorbidity_2020-3-16.txt\"))\n\ncomorb[1:6,1:10]\n\n#> ID A00-A09 A15-A19 A20-A28 A30-A49 A50-A64 A65-A69 A70-A74 A75-A79 A80-A89\n\n#> 1 1 1 0 0 1 0 0 0 0 0\n\n#> 2 10 0 0 0 0 0 0 0 0 0\n\n#> 3 100 0 0 0 0 0 0 0 0 0\n\n#> 4 1000 0 0 0 0 0 0 0 0 0\n\n#> 5 101 0 0 0 0 0 0 0 0 0\n\n#> 6 102 0 0 0 0 0 0 0 0 0\n\nPerforming association tests between COVID-19 phenotype and comorbidities. This is an example of association tests between COVID-19 susceptibility and all comorbidities. It shows NAs when fitted probabilities numerically 0 or 1 occurred in the logistic regression models.\n\ncomorb.asso <- comorbidity.asso (pheno=phe,\n\ncovariates=covar,\n\ncormorbidity=comorb,\n\npopulation=\"white\",\n\ncov.name=c(\"sex\",\"age\",\"bmi\",\"SES\",\"smoke\",\"inAgedCare\"),\n\nphe.name=\"pos.neg\",\n\nICD10.file=covid_example(\"ICD10.coding19.txt.gz\"),\n\noutput = \"cormorb_pos_neg_asso.csv\")\n\nhead (comorb.asso, 4)\n\n#> ICD10 Estimate OR 2.5% 97.5% p\n\n#> A00-A09 A00-A09 Intestinal infectious diseases 0.4722864 1.603657 0.756784 3.240022 0.199664372\n\n#> A15-A19 A15-A19 Tuberculosis NA NA NA NA NA\n\n#> A20-A28 A20-A28 Certain zoonotic bacterial diseases NA NA NA NA NA\n\n#> A30-A49 A30-A49 Other bacterial diseases 1.2246077 3.402831 1.633209 6.978689 0.000873076\n\n\nDiscussion\n\nWe developed an R package that can reproducibly analyse and produce input files for GWAS studies for COVID-19 traits, using the UKBB resource.\n\nThe R package can be easily applied to the frequently updated UKBB COVID-19 datasets, facilitating rapid analyses. By applying the R package to data released in April 2021, we found that age, BMI, SES and smoking are positively associated with COVID-19 susceptibility, severity and mortality. Males are at a higher risk of COVID-19 infection than females. People residing in aged care homes were also at higher risk, potentially because they have other pre-existing conditions, and may also have a higher chance of exposure to SARS-CoV-2. By performing GWAS, we replicated previous findings (Pairo-Castineira et al., 2021; Zeberg and Pääbo, 2020; “Genomewide Association Study of Severe Covid-19 with Respiratory Failure”, 2020; Host Genetics Initiative, 2021) that the locus 3p21.31 is associated with COVID-19 susceptibility and severity.\n\nThe COVID-19 Host Genetics Initiative brings together the human genetics community to generate, share, and analyse data to learn the genetic determinants of COVID-19 susceptibility, severity, and related outcomes. They have been performing large-scale meta-analyses using existing biobanks, including UKBB, and periodically provide updated releases of their results, making available genome-wide summary statistics, and providing an online browser for exploring the latest results (https://app.covid19hg.org/). We primarily advocate the use of these resources for exploring genetic associations with COVID-19 susceptibility and severity. However, we anticipate our R package will enable researchers to undertake more bespoke genetic analyses, using the most up to date UKBB COVID-19 data, to meet the aim of their studies. Such analyses may include adjusting for non-genetic risk factors or comorbidities, to explore mediators, polygenic risk score analyses, or Mendelian Randomisation studies.\n\nThere are several limitations of UKBB COVID-19 data. First, UKBB is not a nationally or worldwide representative sample. The majority of participants are of white British ethnicity. UKBB participants were more likely to be older, to be female, and to live in less socioeconomically deprived areas than nonparticipants. Compared with the general population, participants were less likely to be obese, to smoke, and to drink alcohol daily and had fewer self-reported health conditions (Fry et al., 2017). Initiatives such as OpenSafely (Williamson et al., 2020), have aimed to examine risk factors for COVID-19 disease in an unascertained UK population, via electronic health records. These data, however, are not presently available for use by the wider research community, due to the possibility of re-identification of individuals. The recent OpenSafely flagship paper examined health records of over 17 million individuals in England, of whom 10,926 had a COVID-19 related death, and found that male sex, greater age and deprivation, and non-white ethnicities were major clinical risk factors for mortality. Despite the ascertainment of the UKBB, it is reassuring that these established risk factors are also associated with COVID-19 outcomes in this cohort.\n\nSecond, the UKBB COVID-19 dataset evolved as testing scaled up in line with the national testing strategy and thus COVID-19 data is also subject to ascertainment bias. UK testing was initially largely restricted to healthcare workers, and those individuals with symptoms in hospitals. A positive result in an individual not recorded as a healthcare worker was therefore a reasonable proxy for severe disease early on in the pandemic. Testing capacity subsequently increased to include more community testing under pillar 2 of the national strategy, and as of 27 April 2020, NHS England directed hospitals to test all non-elective patients admitted overnight, including asymptomatic patients. To maximise ascertainment of cases and to evaluate disease severity, SARS-CoV-2 testing data should be used in combination with linked medical records (i.e. hospital inpatient records and death records) as we have implemented in this package. More recently, UKBB has made primary care records available for COVID-19 research. These data not yet utilised by the UKB.COVID19 package, will further improve case identification. Nonetheless, there are likely to be many individuals in the UKBB who contracted COVID-19, in particular those with milder disease, who will not be captured by the available data.\n\nThe definition of COVID-19 susceptibility is supposed to be the status of people who get infected or not after exposure to SARS-CoV-2. However, exposure to SARS-CoV-2 is not easy to determine. Furthermore, not everyone has an equal chance of being exposed to SARS-CoV-2 (for example, exposure will vary by occupation), nor does everyone have the same likelihood of being tested, due to testing strategies, as noted above. Such data idiosyncrasies have the potential to distort associations, in observational studies, and also in genetic analyses through population stratification. This issue of ascertainment, or collider bias, in the context of COVID-19, is discussed at length by Griffith et al., 2020. Analyses using the UKBB data should therefore be undertaken and interpreted within the context of changing testing capacity, and other limitations regarding phenotype definitions.\n\nWe welcome further suggestions and improvements for this R package, which we hope will reduce the barrier to utilising the UKBB data for COVID-19 research.\n\n\nData availability\n\nAll the datasets were obtained from UKBB.\n\nTo access the UKBB datasets, you need to register as a UKBB researcher (https://www.ukbiobank.ac.uk/enable-your-research/register). If you are already an approved UKBB researcher with a project underway and wish to receive these datasets for COVID-19 research purposes, you can register to receive these data by logging into the Access Management System (AMS) (https://bbams.ndph.ox.ac.uk/ams/resApplications).\n\nHow to apply for access to UKBB data: https://www.ukbiobank.ac.uk/enable-your-research/apply-for-access. See COVID-19 data (https://biobank.ndph.ox.ac.uk/showcase/exinfo.cgi?src=COVID19) for registration and access details and Resource 1758 (https://biobank.ndph.ox.ac.uk/showcase/refer.cgi?id=1758) for further information.\n\nAll genome wide significant GWAS hits with gene annotations are shown in Table 6.\n\n\nSoftware availability\n\nUKB.COVID19 can be installed via CRAN using install.packages (“UKB.COVID19”).\n\nUKB.COVID19 is maintained at https://github.com/bahlolab/UKB.COVID19.\n\nLatest UKB.COVID19 source code is available from: https://github.com/bahlolab/UKB.COVID19.\n\nArchived source code at the time of publication: http://doi.org/10.5281/zenodo.5174381 (Wang et al., 2021).\n\nLicense: MIT (https://opensource.org/licenses/MIT).", "appendix": "Acknowledgements\n\nThis research was conducted using data from UK Biobank (www.ukbiobank.ac.uk), a major biomedical database.\n\n\nReferences\n\nBlack D: “HEALTH AND DEPRIVATION: Inequality and the North.” J Royal College General Practitioners. 1988; 38(310):234.\n\nBooth A, Reed AB, Ponzo S, et al.: Population Risk Factors for Severe Disease and Mortality in COVID-19: A Global Systematic Review and Meta-Analysis. PloS One . 2021; 16(3): e0247461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBycroft C, Freeman C, Petkova D, et al.: The UK Biobank Resource with Deep Phenotyping and Genomic Data. Nature. 2018; 562(7726):203–209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElhabyan A, Saja E, Ehab S, et al.: The Role of Host Genetics in Susceptibility to Severe Viral Infections in Humans and Insights into Host Genetics of Severe COVID-19: A Systematic Review. Virus Res. 2020; 289(November): 198163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElliott LT, Sharp K, Alfaro-Almagro F, et al.: Genome-Wide Association Studies of Brain Imaging Phenotypes in UK Biobank. Nature. 2018; 562(7726):210–216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmrani S, Arain HA, DeMarshall C, et al.: APOE4 Is Associated with Cognitive and Pathological Heterogeneity in Patients with Alzheimer’s Disease: A Systematic Review. Alzheimers Res Ther. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFry A, Littlejohns TJ, Sudlow C, et al.: Comparison of Sociodemographic and Health-Related Characteristics of UK Biobank Participants With Those of the General Population. Am J Epidemiol. 2017; 186(9):1026–1034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGenomewide Association Study of Severe Covid-19 with Respiratory Failure. New Eng J Med. 2020; 383(16):1522–1534. Publisher Full Text\n\nGriffith GJ, Morris TT, Tudball MJ, et al.: Collider Bias Undermines Our Understanding of COVID-19 Disease Risk and Severity. Nat Commun. 2020; 11(1): 5749. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann M, Kleine-Weber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHost Genetics Initiative, Covid-19: Mapping the Human Genetic Architecture of COVID-19 by Worldwide Meta-Analysis. MedRxiv. 2021; Reference Source\n\nJiang H, Promchan K, Lin B-R, et al.: LZTFL1 Upregulated by All-Trans Retinoic Acid during CD4+ T Cell Activation Enhances IL-5 Production. J Immunol. 2016; 196(3):1081–1090. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaser A: Genetic Risk of Severe Covid-19. New England J Med. 2020. Publisher Full Text\n\nKuo C-L, Pilling LC, Atkins JL, et al.: ApoE e4e4 Genotype and Mortality With COVID-19 in UK Biobank. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2020a; 75(9):1801–1803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuo C-L, Pilling LC, Atkins JL, et al.: APOE e4 Genotype Predicts Severe COVID-19 in the UK Biobank Community Cohort. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2020b; 75(11):2231–2232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C-C, Liu C-C, Kanekiyo T, et al.: Apolipoprotein E and Alzheimer Disease: Risk, Mechanisms and Therapy. Nat Rev. Neurol 2013; 9 (2): 106–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNumbers K, Brodaty H: The Effects of the COVID-19 Pandemic on People with Dementia. Nat Rev. Neurol. 2021; 17(2):69–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPairo-Castineira E, Clohisey S, Klaric L, et al.: Genetic mechanisms of critical illness in COVID-19. Nature. 2021; 591:92–98. Publisher Full Text\n\nPeckham H, de Gruijter NM , Raine C, et al.: Male Sex Identified by Global COVID-19 Meta-Analysis as a Risk Factor for Death and ITU Admission. Nat Commun. 2020; 11 (1): 6317. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPijls BG, Jolani S, Atherley A, et al.: Demographic Risk Factors for COVID-19 Infection, Severity, ICU Admission and Death: A Meta-Analysis of 59 Studies. BMJ Open. 2021; 11(1): e044640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPurcell S, Neale B, Todd-Brown K, et al.: PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am J Hum Genet. 2007; 81(3):559–575. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSafieh M, Korczyn AD, Michaelson DM: ApoE4: An Emerging Therapeutic Target for Alzheimer’s Disease. BMC Med. 2019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeo S, Zhang Q, Bugge K, et al.: A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened. PLoS Genet. 2011; 7(11): e1002358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang L, Jackson VE, Fearnley LG, et al.: UKB.COVID19: an R package for UK Biobank COVID-19 data processing and analysis. Zenodo. 2021. Publisher Full Text\n\nWilliamson EJ, Walker AJ, Bhaskaran K, et al.: Factors Associated with COVID-19-Related Death Using OpenSAFELY. Nature. 2020; 584(7821):430–436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolff D, Nee S, Hickey NS, et al.: Risk Factors for Covid-19 Severity and Fatality: A Structured Literature Review. Infection. 2021; 49(1):15–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Z, McGoogan JM: Characteristics of and Important Lessons from the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases from the Chinese Center for Disease Control and Prevention. JAMA. 2020; 323(13):1239–1242. PubMed Abstract \| Publisher Full Text\n\nZeberg H, Pääbo S: The Major Genetic Risk Factor for Severe COVID-19 Is Inherited from Neanderthals. Nature. 2020; 587(7835):610–612. PubMed Abstract \| Publisher Full Text\n\nZhao J, Yang Y, Huang H, et al.: Relationship between the ABO Blood Group and the COVID-19 Susceptibility. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. August 2020. Publisher Full Text\n\nZhou W, Nielsen JB, Fritsche LG, et al.: Efficiently Controlling for Case-Control Imbalance and Sample Relatedness in Large-Scale Genetic Association Studies. Nat Genet. 2018; 50 (9). Publisher Full Text\n\nZietz M, Zucker J, Tatonetti NP: Associations between Blood Type and COVID-19 Infection, Intubation, and Death. Nat Commun. 2020; 11(1): 5761. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "100445", "date": "02 Dec 2021", "name": "Thomas Michael Palmer", "expertise": [ "Reviewer Expertise Medical Statistics / Biostatistics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBefore I review this R package properly there are some basic fixes to the GitHub repository version which require attention.\nThe package has an unusual history. Two versions have been released on CRAN however as I can see from the website it was “Archived on 2021-10-06 as email to the maintainer was undeliverable”. So I recommend that the authors contact CRAN to get the package unarchived.\n\nThe CRAN archive shows versions 0.1.0 and 0.1.1, however the GitHub repo shows version 0.1.0 in its DESCRIPTION file. The repo should have the latest version in it.\n\nWhilst the versions listed on CRAN 0.1.0 and 0.1.1 must have been CRAN compliant, otherwise they would not have been allowed on CRAN, unfortunately the code in the GitHub is no longer CRAN compliant and a simple running of R CMD check on the code in the repo gives 2 R CMD check errors and 1 note. These R CMD check errors should be fixed and the R CMD check note should also be fixed by adding the relevant entries to the .Rbuildignore file.\n\nThe script in the tests/testthat folder does not use any of the testthat functions as it should. This should be improved or removed.\n\nPersonally I find the name of the package unusual, I don’t prefer full-stops/periods in package names.\n\nReturned objects from the functions could be defined under one of the R's class systems, e.g., S3.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "7662", "date": "10 Jan 2022", "name": "Longfei Wang", "role": "Author Response", "response": "1.The package has an unusual history. Two versions have been released on CRAN however as I can see from the website it was “Archived on 2021-10-06 as email to the maintainer was undeliverable”. So I recommend that the authors contact CRAN to get the package unarchived. My apologies that I was not aware that the package had been archived. I have contacted the CRAN team. They replied that a CRAN team member tried to contact me and the email has got a bounced message notification. However, my email address is correct and has not been changed since I submitted the package. I have resubmitted the package with an increased version number and with minor changes according to your suggestions. It has been unarchived (https://cran.r-project.org/web/packages/UKB.COVID19/index.html). 2. The CRAN archive shows versions 0.1.0 and 0.1.1, however the GitHub repo shows version 0.1.0 in its DESCRIPTION file. The repo should have the latest version in it. My apologies that the package on GitHub was out-of-date. I have updated the latest version in GitHub. 3. Whilst the versions listed on CRAN 0.1.0 and 0.1.1 must have been CRAN compliant, otherwise they would not have been allowed on CRAN, unfortunately the code in the GitHub is no longer CRAN compliant and a simple running of R CMD check on the code in the repo gives 2 R CMD check errors and 1 note. These R CMD check errors should be fixed and the R CMD check note should also be fixed by adding the relevant entries to the .Rbuildignore file. I have updated the latest version in GitHub and double checked it with R CMD check. There’s no errors, warnings, or notes from the R CMD check now. 4. The script in the tests/testthat folder does not use any of the testthat functions as it should. This should be improved or removed. Thanks for your suggestion. I have improved the scripts in the tests/testthat folder with proper testthat functions. 5. Personally I find the name of the package unusual, I don’t prefer full-stops/periods in package names. Thanks for your suggestion. The package has been on CRAN for a while. People may have included the package in their scripts. These scripts will break if I change the name of the package. And it may be hard for everyone to find the renamed package. So I decided to keep the name and will definitely use proper names for the packages I build in the future. 6. Returned objects from the functions could be defined under one of the R's class systems, e.g., S3. Thanks for your suggestion. I have defined the returned objects under the S3 class system." } ] }, { "id": "126903", "date": "22 Apr 2022", "name": "Virginia Valeria", "expertise": [ "Reviewer Expertise biostatistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors developed a potentially useful R-package tool to analyze data from the UKBB COVID-19 database, which summarises COVID-19 test results, and performs association tests between COVID-19 susceptibility/severity and potential risk factors such as age, sex, blood type, comorbidities and generates input files for GWAS.\nThe rationale is well explained, sufficient details of the code, methods, and analysis are provided, outputs are well described and conclusions are sound and appropriate.\nHowever, some minor points should be considered:\nIt is not clear how comorbidities are retrieved, classified (at which level of ICD-10), and analysed\n\nAuthors should discuss how they choose to classify severity (the distinction between critical care and advanced critical care for example) and why they choose to include all Covid patients (for example severity 2-3 vs 0-1 instead of severity 2-3 vs 1). Why not consider it as an ordinal variable?\n\nAuthors should specify if they consider mortality due to Covid or with Covid\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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": "8179", "date": "18 May 2022", "name": "Longfei Wang", "role": "Author Response", "response": "1. It is not clear how comorbidities are retrieved, classified (at which level of ICD-10), and analysed. We added a new table (Table 4) to show how the comorbidities are classified. We modified the following sentences: Comorbidity categories are generated using the block categories in the ICD10 code, which is shown in the second column in Table 4. We include ICD10 chapters 1–14 and 17 and exclude several chapters such as pregnancy, childbirth, and consequences of external causes etc. For instance, the first category is “A00-A09”, representing intestinal infectious diseases. During a period restricted by the start and end dates, cases are defined as any participants who were diagnosed as any subclasses under the block A00-A09 in the hospital inpatient diagnosis data. In this way, 164 binary variables are generated and each of them represents a comorbidity category. Therefore, we can test the association between each comorbidity category and the selected COVID-19 phenotype using logistic regression models. We modified the sentence as follows: The comorbidity.asso function performs association tests between each comorbidity category and the selected phenotype using logistic regression models and adjusts the tested phenotype with covariates, which can be set using the argument “cov.name”. 2. Authors should discuss how they choose to classify severity (the distinction between critical care and advanced critical care for example) and why they choose to include all Covid patients (for example severity 2-3 vs 0-1 instead of severity 2-3 vs 1). Why not consider it as an ordinal variable? Thanks for the suggestion. We re-wrote the following paragraph: Based on the World Health Organization (WHO) ordinal scale for clinical improvement, we classify severity into four levels. These levels are defined as 1) hospitalisation: individuals admitted to hospital with their primary diagnosis recorded as COVID-19. 2) critical care level 2: individuals required basic treatment in a critical care unit, such as non-invasive ventilation and continuous positive airway pressure, and with their primary diagnosis recorded as COVID-19. 3) critical care level 3: individuals required advanced treatment in a critical care unit, such as invasive ventilation and temporary tracheostomy, and with their primary diagnosis recorded as COVID-19. 4) mortality: individuals died due to COVID-19. The critical care information was summarised from the HESIN_CRITICAL table and the HESIN_OPER table. The critical care level 2 cases are the COVID-19 patients who required at least one “Critical care level 2 days” in the HESIN_CRIRICAL table or received basic respiratory support, such as, E85.2 non-invasive ventilation NEC, in the HESIN_OPER table. The critical care level 3 cases are defined as the COVID-19 patients who required at least one “Critical care level 3 days” in the HESIN_CRIRICAL table or received advanced respiratory support, such as, E85.1 invasive ventilation, in the HESIN_OPER table. The commonly used GWAS tools, such as SAIGE and PLINK, do not support ordinal categorical phenotypes. Therefore, we converted this ordinal variable into four binary variables named “hospitalisation”, “critical care”, “advanced critical care” and “mortality” (Table 2). However, users can get the ordinal variable by simply summing the four binary variables. We assume that participants who were tested COVID-19 positive but did not admit to hospital had no or mild symptoms and hence classified them as controls in severity phenotypes. 3. Authors should specify if they consider mortality due to Covid or with Covid Sorry for the unclearness. We defined the mortality case as mortality due to Covid. In the article, we wrote: For mortality, we include all individuals who received at least one positive test result and define those whose primary cause of death is recorded as being due to COVID-19 as cases. To make it clearer, we corrected the definition of mortality in Table 2 from “1 = death with COVID-19” to “1 = death due to COVID-19”." } ] } ]	1	https://f1000research.com/articles/10-830
https://f1000research.com/articles/12-160/v1	10 Feb 23	{ "type": "Research Article", "title": "Physicochemical characteristics of Pangasius sp. skin-gelatin-based-edible film enriched with silver nanoparticles", "authors": [ "Rahmi Nurdiani", "Asep A. Prihanto", "Muhamad Firdaus", "Faridatul Nur Aini", "Fajrin Adin Nabilah", "Rosnita A Talib", "Nurul Huda", "Asep A. Prihanto", "Muhamad Firdaus", "Faridatul Nur Aini", "Fajrin Adin Nabilah", "Rosnita A Talib", "Nurul Huda" ], "abstract": "Background: Edible films intended for food packaging have been produced from hydrocolloids, lipids, resins, and composites, including gelatin. Gelatin is known to have a good filming ability and has been suggested as an alternative to non-biodegradable plastics. Naturally active compounds incorporated into film packaging may not only protect the food product from oxidation and microbial contamination, but they may also alter the physicochemical properties of the film. Silver nanoparticles have been used in food packaging as active agents due to their antibacterial and antifungal properties. The addition may affect the characteristics of the packaging. Therefore, this study aims to determine the effect of the addition of silver nanoparticles on the physicochemical characteristics of edible film from Pangasius sp. skin gelatin Methods: Mangrove extract of Bruguiera gymnorrhiza was used to synthesize silver into nanoparticle size. In this study, silver nanoparticles (AgNPs) with different concentrations (0, 2, 4 and 8%) were added into gelatin-based edible film. The edible films produced were observed for their physicochemical characteristics, including thickness, tensile strength, elongation, water vapour transmission, moisture content, pH, and colour. Results: AgNPs affected the colour of the fish-gelatin-based edible film, as an increased concentration of AgNPs resulted in a darker film. Nevertheless, the addition of AgNPs showed no significant effect on the thickness (145–216 µm), tensile strength (14.58–19.72 MPa), elongation (21.86–54.19%), water vapour transmission (30. 91–42.55 g/m2/day), moisture content (9.57–11.16%) or pH (5.92–6.01) of the fish-gelatin-based edible film. Conclusions: The addition of AgNPs has no significant effect on gelatin-based edible film physicochemical properties except colour. Therefore, the incorporated edible film has the potential to be developed further.", "keywords": [ "Bruguiera gymnorrhiza", "edible film", "gelatin", "physicochemical characteristics", "silver nanoparticles" ], "content": "Introduction\n\nDue to their excellent structural properties and performances, many petrochemical-based plastics are widely used. However, since these materials are not deemed environmentally friendly, increased public awareness has led to the continued development of biodegradable packaging technology.1 Edible film, one of the biodegradable packaging materials, is a thin layer attached to a food product and can be consumed.2 Edible films can be made from various materials such as polysaccharides (e.g.: starch, pectins, chitin and chitosan), lipids (e.g.: bees wax, oils), and protein (e.g.: whey, soy, pea protein, collagen, gelatin).\n\nGelatin is a protein macromolecule obtained from the hydrolysis of natural collagen found in skin, bone and animal connective tissue.3 Usually, gelatin comes from cows or pigs, but using raw material sources for gelatin from these animals may cause some problems related to health and religious regulations. Alternatively, fish skin is considered a safer option as a raw material for gelatin production.4 Pangasius catfish skin is a by-product resulting from the processing of fish fillets. The processing of Pangasius catfish fillets produces up to 55% of by-products, including fish skin.5 The skin can potentially be used as a raw material for gelatin due to its abundant availability. Fish gelatin is now commonly used in the production of edible films due to its low melting temperature, low oxygen permeability, and good film-forming ability.6\n\nNowadays, the concept of active packaging has attracted the food packaging industry and researchers. Active packaging may enable the interaction between the packaging, the food product, and the surrounding environment to improve the sensory properties and safety of food products. In addition, active agents with antimicrobial and antioxidant properties can help in extending the food’s shelf life and prevent food oxidation.7 The edible film made using fish skin gelatin usually has low biological properties, so enriching it with active compounds is necessary to produce active packaging. Several studies have shown that edible films made from fish gelatin can be manufactured by adding several active ingredients.8 One group of active ingredients that have the potential to be added are silver nanoparticles. Silver nanoparticles (AgNPs) are materials with at least one dimension with a size between 1 and 100 nm.9 They can be made by synthesizing silver metal into nano-sized particles. This synthesis can be carried out with a biosynthesis biological agent.9 In previous studies, the synthesis of silver nanoparticles from mangrove leaves such as Excoecaria agallocha L.10 and Rhizophora mucronata11,12 has been achieved. In this study, the biosynthesis process was done using the extract of mangrove leaves, Bruguiera gymnorrhiza. The content of bioactive compounds in plant extracts can be used as a bio-reductant that can convert silver metal into AgNPs.9\n\nNurdiani et al.,13 stated that AgNPs can improve the characteristics and functionalities of edible films, such as increasing product resistance, improving barrier properties as packaging and antimicrobial compounds. Therefore, this research was conducted to determine the physicochemical characteristics of edible film from Pangasius catfish skin gelatin with the addition of AgNPs of Bruguiera gymnorrhiza.\n\n\nMethods\n\nPangasius sp. skin (frozen) was provided by PT. RUM Seafood, Sidoarjo Indonesia, and delivered to the Faculty of Fisheries and Marine Science, Universitas Brawijaya. Once the skins were thawed, they were cut into small pieces (0.5 × 0.5 cm2) using clean scissors. The skins were stored in a freezer (−20°C) until use. Gelatin manufacturing started by soaking the Pangasius sp. skin in NaOH 0.1 M solution with a ratio of 1:5 (w/v) for two hours at room temperature. Afterwards, the skin was washed with running water until the pH was normal (pH = 7). Next, the skin was then soaked in an acetic acid solution with a concentration of 0.6 M at a ratio of 1:5 (w/v) for two hours (at room temperature). The swollen skin was then extracted with Aqua Dest, with a ratio of 1:3 (w/v) in a water bath (at 55–60°C) for four hours. Afterwards, the filtrate was collected and poured onto a baking tray for drying using a dehydrator for 8 to 12 hours at 55–60°C. The gelatin sheets obtained were then milled using a grinder to make gelatin powder.8\n\nThe leaves of Bruguiera gymnorrhiza were obtained from the Sendang Biru coastal area, Malang, East Java, Indonesia. The leaves were dried using a dehydrator for 8–12 hours at a temperature of 40–45°C. Dried leaves were then reduced in size using a grinder. Two grams of mangrove powder was then dissolved into 100 mL of deionized water and heated to boiling for three minutes. The mangrove solution was allowed to cool to room temperature, filtered using a Buchner funnel, and poured into a dark glass bottle.10\n\nOne mL of mangrove leaf extract was mixed with 9 mL 20 mM AgNO3. The solutions were incubated for 0, 5, 10, and 15 minutes. The colour change was observed from the original whitish brown to brown.9\n\nEdible film was made by dissolving 4 grams gelatin into 100 mL distilled water. The gelatin solution was then heated at 50°C for 30 minutes. Next, the glycerol was added at 0.5% (v/v) and heated at 45°C for 15 minutes. AgNPs with the best incubation time was added at several concentrations per 100 mL (0 mL, 2 mL, 4 mL, and 8 mL). Once ready, the edible film solution was evenly poured into an 18 cm × 18 cm non-stick baking pan before being dried in an oven at a temperature of 55°C for 18–20 hours. The edible film was then removed from the oven, left at room temperature for 10 minutes, and peeled off slowly.13\n\nThickness\n\nThe thickness of the edible film sample was measured using a micrometre screw with an accuracy of 0.01 mm. Thickness measurements were carried out at five different points, and the average results represented the sample.14\n\nMoisture content\n\nAn empty crucible cup was oven dried at 105°C for 30 minutes. As it cooled down, 1 gram of the sample was then put into the cup and weighed (W1). Next, the sample was put into the oven and dried at a temperature of 105°C for three hours. After that, the cup was weighed again (W2).15\n\nWater vapour transmission rate (WVTR)\n\nThe WVTR of each film was measured according to Ref. 16. The edible film was cut into a circle with a diameter of 3 cm. Ten grams of silica gel were poured into a cup. The sample was glued on top of the cup to cover it. The cup was weighed and recorded as W1. The sample was allowed to stand for 24 hours at room temperature and was then weighed again and recorded as W2. The WVTR was then calculated using the formula:\n\nTensile strength and elongation\n\nEdible film was cut to a size of 5 × 1 cm2. The thickness and initial length of the sample were measured. Next, each end of the edible film was clamped on a tensile machine. The sample was then pulled at 10 mm/min speed until it broke.14 Finally, the tensile strength value was calculated by dividing the maximum stress (F max) by the area of the edible film using the formula:\n\nThe measurement of the elongation of the edible film was carried out using a tensile strength device. Elongation is expressed as a percentage and is measured using the formula:\n\npH\n\nThe pH test was carried out using a pH meter. The measurement was conducted on the edible film solution prior to the drying process.17\n\nColour\n\nThe colour of the edible films was measured using a colorimeter. Measurements were made by placing an edible film sample on the colour reader sensor. The reading button was set to L* (lightness), a* (redness) and b* (yellowness), and the target button was pressed. The results then appeared on the screen.\n\nSPSS commercial software (IBM SPSS Statistics version 25.0, Chicago, IL, USA) was used for statistical analysis. The data were expressed as mean ± standard deviation (mean ± SD). A one-way analysis of variance (ANOVA) and Tukey’s test with a p < 0.05 significance level were applied.\n\n\nResults and discussion\n\nThickness is one of the characteristics of edible coatings that greatly affects the biological properties and shelf life of food-coating products.18 This study showed that the addition of AgNPs into the gelatin solution had no significant effect (p > 0.05) on the thickness of edible film. The average thickness of the edible film was between 145 and 216 μm (Table 1). According to the Japanese Industrial Standard (JIS),19 the maximum thickness of edible film should be 250 μm. The thickness of the edible film is affected by the density, viscosity and surface tension of edible film materials as well as the drying time and layering technique.20\n\nSimilarly, Arfat et al.19 reported an increasing of edible film thickness due to the addition of silver–copper nanoparticles (Ag–CuNPs). The resulting range was 61–98 μm. The increase in thickness of the edible film was related to the increase of the solid content of the edible film. Furthermore, the addition of Ag–CuNPs to the gelatin affected the structure of the protein, causing it to become regular and the film network to be stronger.\n\nThe moisture content test was crucial to determine the total number of water molecules in the edible film tissue. The moisture content in the edible film affects the stability of the product, so it is expected that the moisture content in the edible film produced is low. This study showed that adding AgNPs into the gelatin solution had no significant effect (p > 0.05) on the moisture content of each edible film. The moisture content of the edible film had an average value of between 9.57 and 11.16% (Table 1). Our result was lower than the study reported by Kanmani & Rhim.21\n\nAdding AgNPs into an edible film will usually increase its moisture content because of reduced interactions between gelatin chains and increased availability of free hydroxyl groups to absorb water. The moisture content of edible films can also be affected by temperature and drying methods. The temperature will affect the time needed in the drying process, causing an increase in the intensity of the interaction between water molecules and the hydrophilic polymer chain.22\n\nStatistically, the WVTR test indicated that adding AgNPs into the gelatin solution had no significant effect (p > 0.05) on the value of the WVTR of the edible film. The average value of the WVTR of the edible film produced was 30.91–42.55 g/m2/day (Table 1). Our results did not meet the Japanese Industrial Standard (JIS),23 a maximum WVTR of 7 g/m2/day. The high value of the WVTR of the edible films is due to the fact that the materials used to make the edibles are made of protein-derived materials that have polar polymers and a large number of hydrogen bonds, thus making the edible films capable of absorbing water at high humidity. The factors that can affect the WVTR values are the presence of chemicals and the structure of the constituent materials, the concentration of plasticizers, and environmental conditions such as humidity and temperature.24\n\nTable 1 shows that adding AgNPs into the gelatin-based edible film solution had no significant effect (p > 0.05) on the tensile strength of the edible film. The average tensile strength of the edible film was 14.58–19.72 MPa (Table 1). The results are in accordance with the Japanese Industrial Standard (JIS),23 as the minimum tensile strength of edible film should be 0.3 MPa. The value of the tensile strength of the edible films decreased with increasing concentrations of AgNPs. This could be because AgNPs are very weak in forming crosslinks with fish skin gelatin during the production of the edible films. In comparison, the elongation test results showed that adding AgNPs into the gelatin solution had no significant effect (p > 0.05) on the elongation of each edible film. The resulting edible film elongation value ranged from 54.19 to 21.86% (Table 1). The results, however, were still much lower than the standard minimum elongation value of 70.23 Elongation is inversely proportional to tensile strength.18 According to Skurtys et al.,25 elongation is expressed as a percentage change in the film’s original length before breaking and gives a measure of film elongation. Elongation values range from 1 to 80%. The level of elasticity of the edible coating can be seen from the elongation value; the higher the elongation value, the more elastic the edible coating.\n\nThe addition of AgNPs into the film resulted in a decrease in the tensile strength value. This might be due to the reduction in protein chain reactions due to incorporation with AgNPs.21 The tensile strength of a film can be influenced by the constituent materials that have structural cohesion properties and the constituent materials of edible films, namely hydrocolloid protein (gelatin), contain amino acids capable of forming disulphide bonds.26 In addition, the properties of the constituent materials to create a more robust gel network will help the formation of polysaccharide molecular structures more closely, forming a more coherent film structure and reducing the absorption of water molecules.27 Bonilla & Sobral28 stated that the elongation value of gelatin and chitosan edible films with ethanolic extracts from plants increased with the increased volume of extracts. This is due to the interaction between the phenolic compounds present in the extract. Furthermore, gelatin peptides can form covalent crosslinks, forming a more cohesive and flexible matrix. The higher the elongation value of the film, the more elastic it is so that the film can be stretched more. Meanwhile, films with low elongation values will be brittle.\n\nThe results showed that the pH of the enriched edible film ranged from 5.92 to 6.01 (Table 1). It was indicated that the higher the concentration of AgNPs Bruguiera gymnorrhiza added, the lower the pH level of fish-gelatin-based edible film. Bruguiera gymnorrhiza mangrove leaf extract was detected to have an acidic pH of around 6.15, thus, the resulting edible film was expected to have a lower pH value than the control (0% AgNPs).\n\nColour testing using a colour reader brings up three values: L, a, and b. The L value indicates the brightness level, a* indicates a red–green colour (a = +60 red, a = -60 green) and b* indicates a yellow–blue colour (b = +60 yellow, b = -60 blue). Edible films produced using gelatin usually have clear or dull/opaque colours.13 Statistically, the addition of the concentration of AgNPs had a significant effect (p < 0.05) on the L, a and b* values (Table 2).\n\nThe addition of a higher concentration of AgNPs on the fish-gelatin-based edible film resulted in a darker colour (Figure 1), increasing the red and yellowish colour. Similarly, Shankar et al.29 reported that the L* value of edible gelatin films with the addition of metallic nanoparticles (38.20) was lower than the control (93.15).\n\nThe best treatment of edible film made from Pangasius skin gelatin with the addition of silver nanoparticles synthesized using Bruguiera gymnorrhiza extract was determined using the De Garmo method (grouping and weighting parameters, where the weights given are in accordance with the importance/priority of each parameter in affecting the results of the research).30 Based on the calculation, it was suggested that the best treatment for all parameters was the third treatment (the addition of 4% silver nanoparticles) (Table 3).\n\n\nConclusion\n\nThe addition of various concentrations of AgNPs (0, 2, 4, and 8%) synthesized using mangrove Bruguiera gymnorrhiza had a significant effect on the colour of Pangasius gelatin-based edible film (L* value 70.773–87.363, a* value 0.440–7.398 and b* value 1.978–17.178). Nevertheless, adding AgNPs did not significantly affect the other physicochemical parameters (thickness, tensile strength, elongation, water vapour transmission, moisture content, and pH). It was suggested to use 4% AgNPs to get the best characteristics of the edible film based on the available standard. Based on the results, it is recommended to use AgNPs as active agents to produce active packaging since they have no unfavourable effect on the physicochemical properties of edible film. Further studies on the biological properties of edible film enriched with AgNPs is necessary.\n\n\nEthical approval\n\nThis study did not require ethical approval because the authors used fish skins (available as waste or by-products) from the fish processing industry. Mangrove leaves were collected from a local mangrove conservation area in Malang, East Java. The conservation officer carried out the identification and collection of the mangrove leaves. The process was conducted in a way to ensures the sustainability of the local mangrove forest.", "appendix": "Data availability\n\nFigshare: Physicochemical Characteristics of Pangasius sp. Skin Gelatin-based Edible Film Enriched with Silver Nanoparticles. https://doi.org/10.6084/m9.figshare.21640196.v2. 32\n\nThe project contains the following underlying data:\n\n− DATA F1000 R NURDIANI.xlsx (raw data for colour, thickness, moisture content, water vapour transmission rate, pH, and tensile strength and elongation).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors would like to thank Universitas Brawijaya and the Ministry of Education, Culture, Research and Technology, the Republic of Indonesia, for the research grant.\n\n\nReferences\n\nMoshood TD, Nawanir G, Mahmud F, et al.: Green product innovation: A means towards achieving global sustainable product within biodegradable plastic industry. J. Clean. Prod. 2022; 363(132506): 132506–132517. Publisher Full Text\n\nKumar N: Neeraj: Polysaccharide-based component and their relevance in edible film/coating: a review. Nutr. Food Sci. 2019; 49(5): 793–823. Publisher Full Text\n\nJaziri AA, Shapawi R, Mokhtar RAM, et al.: Biochemical analysis of collagens from the bone of lizardfish (Saurida tumbil Bloch, 1795) extracted with different acids. PeerJ. 2022; 10(e13103): e13103–e13122. Publisher Full Text\n\nJaziri AA, Muyasyaroh H, Firdaus M: Effect of phosphoric acid concentration on physicochemical properties of Abalistes stellaris skin gelatin. IOP Conf. Ser.: Earth Environ. Sci. 2020; 493(012038): 012037–012038. Publisher Full Text\n\nHastarini E, Nabilla M, Permadi A, et al.: Characteristic of margarine with ingredient mixed of catfish (Pangasius sp.) oil and vegetable oil. IOP Conf. Ser.: Earth Environ. Sci. 2021; 919(012043): 012012–012043. Publisher Full Text\n\nChin SS, Lyn FH, Hanani ZAN: Effect of Aloe vera (Aloe barbadensis Miller) gel on the physical and functional properties of fish gelatin films as active packaging. Food Packag. Shelf Life. 2017; 12: 128–134. Publisher Full Text\n\nRealini CE, Marcos B: Active and intelligent packaging systems for a modern society. Meat Sci. 2014; 98(3): 404–419. PubMed Abstract \| Publisher Full Text\n\nSánchez JT, Vilaplana F, García AV, et al.: Physicochemical and Functional Properties of Active Fish Gelatin-Based Edible Films Added with Aloe Vera Gel. Food. 2020; 9(9): 1–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdi V, Sourinejad I, Yousefzadi M, et al.: Mangrove-mediated synthesis of silver nanoparticles using native Avicennia marina plant extract from southern Iran. Chem. Eng. Commun. 2018; 205(8): 1069–1076. Publisher Full Text\n\nBhuvaneswari R, Xavier RJ, Arumugam M: Facile synthesis of multifunctional silver nanoparticles using mangrove plant Excoecaria agallocha L. for its antibacterial, antioxidant and cytotoxic effects. J. Parasit. Dis. 2017; 41: 180–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUmashankari J, Inbakandan D, Ajithkumar TT, et al.: Mangrove plant, Rhizophora mucronata (Lamk, 1804) mediated one pot green synthesis of silver nanoparticles and its antibacterial activity against aquatic pathogens. Aquatic Biosystems. 2012; 8(11): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdi V, Sourinejad I, Yousefzadi M, et al.: Biosynthesis of Silver Nanoparticles from the Mangrove Rhizophora mucronata: Its Characterization and Antibacterial Potential. Iran. J. Sci. Technol. Trans. A Sci. 2019; 43: 2163–2171. Publisher Full Text\n\nNurdiani R, Prihanto AA, Firdaus M, et al.: Antibacterial and Antioxidant Activities of Edible Film Incorporated with Silver Nanoparticles Synthesized using Rhizophora mucronata Extract. IOP Conf. Ser.: Earth Environ. Sci. 2022; 1118(2022): 012031–012039. Publisher Full Text\n\nChae S, Heo TR: Production and properties of edible film using whey protein. Biotechnol. Bioprocess Eng. 1997; 2: 122–125. Publisher Full Text\n\nAOAC:Official Methods of Analysis. The Association of Official Analitical Chemist. Washington, DC:A. O. A. C. Inc;1995; 38. : 1–3. Chap.\n\nAmerican Society for Testing and Materials (ASTM): Standard test m ethods for water vapor transmission of materials ASTM E. Philadelphia:ASTM;1987; 629–636.\n\nDari DW, Masruroh LA, Junita D: Chemical characteristics and acidity of pedada fruit juice (Sonneratia sp.) with the addition of sodium benzoate. J. Food Technol. Nutr. Sci. 2021; 20(1): 35–44.\n\nHatmi RU, Apriyati E, Cahyaningrum N: Edible Coating Quality with Three Types of Starch and Sorbitol Plasticizer. E3S Web Conf. 2020; 142(02003): 1–9. Publisher Full Text\n\nArfat YA, Ahmed J, Hiremath N, et al.: Thermo-mechanical, rheological, structural and antimicrobial properties of bionanocomposite films based on fish skin gelatin and silver-copper nanoparticles. Food Hydrocoll. 2017; 62: 191–202. Publisher Full Text\n\nCisneros-Zevallos L, Krochta JM: Dependence of Coating Thickness on Viscosity of Coating Solution Applied to Fruits and Vegetables by Dipping Method. J. Food Sci. 2003; 68(2): 503–510. Publisher Full Text\n\nKanmani P, Rhim JW: Physicochemical properties of gelatin/silver nanoparticle antimicrobial composite films. Food Chem. 2014; 148: 162–169. PubMed Abstract \| Publisher Full Text\n\nHomez-Jara A, Daza LD, Aguirre DM, et al.: Characterization of chitosan edible films obtained with various polymer concentrations and drying temperatures. Int. J. Biol. Macromol. 2018; 113: 1233–1240. PubMed Abstract \| Publisher Full Text\n\nJapanese Industrial Standard. Japanese Standards Association;1975; vol. 2. : 1707.\n\nMcHugh TH, Krochta JM: Sorbitol-Gliserol-Plasticized whey protein edible film: integrated oxygen permeability and tensile property evaluation. J. Agric. Food Sci. 1994; 42: 841–845. Publisher Full Text\n\nSkurtys O, Acevedo C, Pedreschi F, et al.: Food hydrocolloid edible films and coatings. Food Hydrocolloid Edible Films and Coatings. 2011; 1–66.\n\nGontard N, Guilbert S, Lug JL: Water and glycerol a plasticizer affect mechanical and water vapor barrier properties of in edible wheat gluten film. J. Food Sci. 1993; 58(1): 206–211. Publisher Full Text\n\nCerqueira MA, Souza BWS, Teixeira JA, et al.: Effects of Interactions between the Constituents of Chitosan-Edible Films on Their Physical Properties. Food Bioprocess Technol. 2012; 5: 3181–3192. Publisher Full Text\n\nBonilla J, Sobral PJA: Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts. Food Biosci. 2016; 16: 17–25. Publisher Full Text\n\nShankar S, Jaiswal L, Selvakannan PR, et al.: Gelatin dissolvable antibacterial films reinforced with metallic nanoparticles. RSC Adv. 2016; 6(71): 67340–67352. Publisher Full Text\n\nDeGarmo EP, Sullivan WG, Canada CR: Engineering Economy. New York:MacMillan Publishing Company;1984.\n\nNurdiani R, Ma’rifah RDA, Busyro IK, et al.: Physical and functional properties of fish gelatin-based film incorporated with mangrove extracts. PeerJ. 2022; 10(e13062): e13062–e13017. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFigshare: Physicochemical Characteristics of Pangasius sp. Skin Gelatin-based Edible Film Enriched with Silver Nanoparticles. DOI: 10.6084/m9.figshare.21640196.v2" }	[ { "id": "208387", "date": "10 Oct 2023", "name": "Mohamed Bilal Goudjil", "expertise": [ "Reviewer Expertise Phytochemistry", "Essential oil", "Chromatography", "Renewable Energies", "Nanotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is interesting original research that aims to investigate the Physicochemical characteristics of Pangasius sp. skin-gelatin-based-edible film enriched with silver nanoparticles.\nOverall, the manuscript is well complied and experimental data is appreciable. However, a few comments below must be addressed by the authors with utmost attention before considering for indexing.\n1. In introduction section: Please give some information about the plant used in the present study.\n2. Author should provide herbarium number or plant code for the Bruguiera gymnorrhiza used.\n3. In the Results and discussion section, a brief talk about the reasons for the formation of nanoparticles using green synthesis, specifying the mechanism and the responsible component, is required.\n4. The conclusion is a bit poor, it should be improved to reflect the work done.\n5. I believe the language can be more polished by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "12021", "date": "25 Jul 2024", "name": "Rahmi Nurdiani", "role": "Author Response", "response": "Comments from the Reviewers 1st REVIEWER Comment 1: In introduction section: Please give some information about the plant used in the present study. Response 1: Bruguiera gymnorhiza, a member of the Rhizophoraceae family, is characterized by its glabrous and relatively smooth trunk with reddish-brown bark. This species is typically found along the seaward margins of mangrove swamps. B. gymnorhiza leaf extract has demonstrated potential as an active compound for incorporation into fish gelatin films, exhibiting inhibitory effects against various pathogenic bacteria such as Pseudomonas aeruginosa (5.77 - 23.13 mm), Aeromonas hydrophila (14.17 - 23.47 mm), and Staphylococcus aureus (1.70 - 4.43 mm) (Effendi et al., 2023). Additionally, the leaf extract has shown significant radical scavenging activity, with an IC50 value of 12.93 µg/mL (Indriaty et al., 2023). Comment 2: Author should provide herbarium number or plant code for the Bruguiera gymnorrhiza used. Response 2: The leaves of Bruguiera gymnorrhiza (Taxonomy ID: 39984) were obtained from the Sendang Biru coastal area, Malang, East Java, Indonesia. Comment 3: In the Results and discussion section, a brief talk about the reasons for the formation of nanoparticles using green synthesis, specifying the mechanism and the responsible component, is required. Response 3: The physicochemical properties of edible films enriched with various concentrations of silver nanoparticles were evaluated. In this study, silver nanoparticles were synthesized using green synthesis with mangrove leaf extract. The reason for the formation of nanoparticles using green synthesis for active agents incorporated in gelatin edible films is biocompatibility. Green-synthesized nanoparticles are often biocompatible and non-toxic, making them suitable for use in edible films that come into direct contact with food products.22 The green synthesis of nanoparticles using plant extracts involves a mechanism that includes the reduction of metal ions, complex formation, stabilization, and shape control. This process utilizes the natural compounds present in plant extracts to facilitate the synthesis of nanoparticles with unique properties.23 Comment 4: The conclusion is a bit poor; it should be improved to reflect the work done. Response 4: The addition of various concentrations of AgNPs (0, 2, 4, and 8%) synthesized using mangrove Bruguiera gymnorrhiza had a significant effect on the colour of Pangasius gelatin-based edible film (L* value 70.773–87.363, a* value 0.440–7.398 and b* value 1.978–17.178). Nevertheless, adding AgNPs did not significantly affect the other physicochemical parameters (thickness, tensile strength, elongation, water vapour transmission, moisture content, and pH). This study recommends using 4% AgNPs to achieve the best qualities in edible film, meeting established standards. The results suggest that AgNPs can be effective active agents for developing active packaging without negatively affecting the film's physicochemical properties. However, further research is needed to explore the biological functions of edible films with added AgNPs. Comment 5: I believe the language can be more polished by the authors. Response 5: Regarding the grammatical and sentence construction errors, we have revised our manuscript." } ] }, { "id": "166792", "date": "28 May 2024", "name": "Amiza Mat Amin", "expertise": [ "Reviewer Expertise food science", "food protein" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 described the effect of incorporating silver nanoparticles in the silver catfish gelatin film. The introduction is sufficient. Please state why the range of 2-8% was used in this study. However, this study did not include antimicrobial activity of the gelatin films. Please comment why there was no significant difference in most results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "12022", "date": "25 Jul 2024", "name": "Rahmi Nurdiani", "role": "Author Response", "response": "Responses to Reviewer 2 Comment 1: Please state why the range of 2-8% was used in this study. However, this study did not include antimicrobial activity of the gelatine films. Response 1: The concentration of silver nanoparticles (AgNPs) added to the edible film ranged from the minimum amount needed to show antibacterial activity to the maximum amount considered safe. Comment 2: Please comment why there was no significant difference in most results. Response 2: The physicochemical properties of edible films enriched with various concentrations of silver nanoparticles were evaluated. The addition of silver nanoparticles, regardless of the concentration, did not significantly affect any of the physicochemical parameters evaluated (Table 1). This might be because the concentration of silver nanoparticles added was very small. Similar to the study conducted by Gutiérrez et al. 24, which investigated the impact of adding calcium and silver nanoparticles to gelatin-based films. They found that the films' structural integrity and mechanical properties were preserved. The dispersion of AgNPs within the gelatin matrix was uniform, preventing significant changes in the film's tensile strength and elasticity. Additionally, the barrier properties, such as water vapour permeability, remained stable." } ] } ]	1	https://f1000research.com/articles/12-160
https://f1000research.com/articles/12-7/v1	04 Jan 23	{ "type": "Case Report", "title": "Case Report: Modified endoscopic hook for extracting magnetic esophageal foreign bodies in a rural area", "authors": [ "Hamsu Kadriyan", "Ika Primayanti", "Syamsidar Syamsidar", "Lalu Fauzan Fakhrussiam", "Moh Fahrur Rozi", "Hijrinelly Hijrinelly", "Ika Primayanti", "Syamsidar Syamsidar", "Lalu Fauzan Fakhrussiam", "Moh Fahrur Rozi", "Hijrinelly Hijrinelly" ], "abstract": "Foreign bodies (FB) in the aerodigestive tract are common, especially in children. The type of foreign body in the esophagus can vary and include magnets. A magnet that lodges in the esophagus should be extracted within 24 hours to prevent complications due to associated chemical reactions. In rural areas, there are several limitations to extracting FBs from the esophagus. We report a case of a magnetic FB that lodged in the esophagus of a three-year-old boy. The extraction was successfully done by esophagoscopy with the modification of a hook that was attached to the endoscope. This innovation may help otolaryngologists all over the world, especially in rural areas. In the future, this innovation could be produced on an industrial scale.", "keywords": [ "Foreign bodies", "aerodigestive tract", "endoscopic hook", "esophagoscopy" ], "content": "Introduction\n\nAlthough a warning regarding choking risk has been included on every toy by the factory,1 cases of foreign body (FB) in the upper aerodigestive tract remain frequently found in the clinical setting. Several types of FBs can be found in the upper aerodigestive tract, including coins, magnets, batteries, pins, and organic substances such as peanuts, meat, among others.2–4 The effect of FBs in the aerodigestive tract varies, depending on its location. If the FB is lodged in the esophagus, it will cause dysphagia, while in the bronchus it may cause airway obstruction and lead to mortality.2,3\n\nThe part of a toy such as a magnet can usually found in children and may cause perforation or fistula on the esophagus. This complication occurs because of the effect of the chemical reaction between the magnet with the esophagus tissue. Therefore, a magnet FB in the esophagus should be extracted within 24 hours of the finding.3,4\n\nThe extraction of FBs from the esophagus may be challenging, especially in a rural area with limited equipment. An endoscope or esophagoscope, and a forceps or extractor are not complete or do not fit with the type or shape of the FBs. Therefore, in rural areas, the physician should try to do their best to help the patient with those limitations. In this report, the authors would like to share the modification of the hook that is attached to the rigid endoscope. This modification successfully extracted a big and thick heart-shaped magnet in the esophagus.\n\n\nCase presentation\n\nA three-year-old boy was referred from the primary hospital with FBs lodged in the esophagus 1 hour prior to the hospitalization. He accidentally ingested the magnet toys while playing with his sister. After the incident, the boy was crying and his sister told their mother that her brother had ingested the toy part. His sister showed the shape of FB that was ingested to their mother. Therefore, the boy was then brought to the hospital to see whether the FB was lodged somewhere or not. There was no sign of coughing or dyspnea in this patient.\n\nAn x-ray examination was done to know the location of the FBs in the upper aerodigestive tract. The result showed the radiopaque metallic object with a heart shape was lodged in the upper esophagus (Figure 1A and B). After the diagnosis was established, the patient was scheduled for an esophagoscopy to extract the FB under general anesthesia.\n\nDuring the esophagoscopy, several forceps and baskets were tried to extract the FB, however, the FB was moving down to the middle part. After several attempts, the authors then tried to modify the endoscope by placing the additional hook on the tip of the rigid scope (Figure 2, red arrow). Finally, the FB could be extracted without any complications, however, the color of the FB has changed (Figure 2, yellow arrow) from its original color (Figure 2, green arrow).\n\nFollow-up after surgery was done after two (2) days, and no complications occurred. The patient was then released from the ward. A week after the surgery, the patient came to the outpatient clinic. No difficulties in swallowing were found, and the patient could eat all kinds of food.\n\n\nDiscussion\n\nThe constraints in managing certain cases in rural areas may be unexpected; therefore, some physicians may refer the patient to a bigger/higher hospital if they encounter difficulties.5 However, in some archipelago countries, the transportation between the islands may become a problem. Some small islands may only have boating infrastructure; on the other hand, larger islands may have a more complete transportation infrastructure. In some cases, the patient or their family may not agree to be referred to another hospital on other islands. Another patient may delay the treatment time for another reason, for instance, costs considerations and low awareness of disease symptoms.6,7 Therefore, the physician should apply their best competencies to manage the cases. One of the general constraints faced in rural areas is inadequate equipment. The other constraint is manpower.7\n\nThe modification of this hook was inspired by the cerumen hook that is routinely used by the otolaryngologist to remove the hard wax or FB in the ear canal.8 Therefore, the principle is alike to a cerumen hook. To make it visible, the hook was attached to the rigid scope. Therefore, the FB extraction could be done safely with direct vision. The other concern is the hook should be in a safe mode for the esophagus, with non-sharp tip for example.\n\nThe extraction itself started with the insertion of a rigid esophagoscope into the esophagus until it was close to the FB location. Then, the hook attached to the scope was inserted through the lumen of the esophagoscope. The hook insertion should be done smoothly in a similar direction to the FB position and esophagus wall (Figure 2) to prevent a wound that may provoke a perforation. After passing the edge of the FB, the hook direction was then rotated to the body of FB and extracted slowly. If it does not work on the first attempt, the procedure could be repeated until the FB is successfully removed.\n\nThe application of a hook is suitable for a quadrangle- or triangle-shape FB with a certain thickness (thicker than a coin). According to a previous publication, a hook for extracting FB in the esophagus is not available.4 Therefore, this modification is the first innovation of the kind published in a journal. Hopefully, this innovation could help otolaryngologists globally, especially in rural areas. This innovation may also be used as an inspiration to modify incomplete equipment in a rural area. However, advanced research should be done to prove it’s efficacy. Therefore, in the future, this modification could be produced on an industrial scale.\n\n\nConclusions\n\nThe modified endoscopic hook is an innovation that is suitable and safe for extracting quadrangle or triangle FB shapes in the esophagus with a certain thickness.\n\n\nConsent of the patient’s parents\n\nThe written consent for using the images and publishing this case report has been given by the parent of the patient.", "appendix": "References\n\nChild Safety Central: Consumer Product Safety Commission: Child Safety Protection Act Fact Sheet.2006.Reference SourceReference Source\n\nGezer HO, Ezer SS, Temiz A, et al.: Ingested foreign bodies in children: Do they really pass spontaneously from the gastrointestinal tract? A single-centre experience with 1000 cases. Ulus. Travma Acil Cerrahi Derg. 2020; 26: 247–254. PubMed Abstract \| Publisher Full Text\n\nAltokhais TI, Al-Saleem A, Gado A, et al.: Esophageal foreign bodies in children: Emphasis on complicated cases. Asian J. Surg. 2017; 40: 362–366. PubMed Abstract \| Publisher Full Text\n\nMagalhães-Costa P, Carvalho L, Rodrigues JP, et al.: Endoscopic Management of Foreign Bodies in the Upper Gastrointestinal Tract: An Evidence-Based Review Article. GE Port. J. Gastroenterol. 2016; 23(3): 142–152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKadriyan H, Sulaksana MA, Yudhanto D, et al.: Subcutaneous hemangioma on nasal dorsum: A case report. J. Med. Case Rep. 2020; 14: 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKadriyan H, Endriani E, Andiwijaya FR, et al.: Upper airway obstruction due to bilateral laryngeal polyp: A challenge of treatment in rural area. Crit. Care Shock. 2021; 2021(July): 212–217.\n\nSoepardi EA: Problem in managing foreign bodies in upper aerodigestive tract. Med. J. Indones. 2002; 11(1): 15–18. Publisher Full Text\n\nWatkins C, McCalla CD: Foreign bodies of the head and neck. Pediatr. Emerg. Med. Rep. 2016; 21(7): 1–15." }	[ { "id": "165757", "date": "15 Nov 2023", "name": "Mingyan Cai", "expertise": [ "Reviewer Expertise Endoscopy" ], "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 described a modified endoscopic hook for extracting magnetic esophageal foreign body. It has some novelty, but such big magnetic foreign body will not always successfully hooked by the small hook. The reproduction of the result is limited in other cases, since the hook itself sometimes won't have enough power to extract the foreign body from the narrowing space of the esophageal entrance.\nGI endoscopy with accessary channels through which can apply a series of accessories has more advantages than this rigid scope.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "10609", "date": "23 Nov 2023", "name": "hamsu kadriyan", "role": "Author Response", "response": "Dear Reviewer Thank you for your comment on my manuscript. The setting of this case was in a rural area. If the case was found in an urban area, I agree to the use of a flexible endoscope. To support the importance of this innovation in rural areas, I have inserted some references in the discussion section" } ] }, { "id": "161310", "date": "15 Nov 2023", "name": "Dian Adi Syahputra", "expertise": [ "Reviewer Expertise Pediatric Surgeon" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA beneficial case report on managing FB in areas with limited resources.\nThere are some comments to improve this case report to make it better for readers.\nThe author should not use the term \"otolaryngologists\" in this case report, especially in the abstract and the last paragraph of the discussion. It is better to use global terms such as doctors or physicians.\n\nIn the discussion section, the author should discuss the anatomy of the esophagus, which results in the retention of the FB in the esophagus to the stomach.\n\nThe author should refrain from doing self-citation in this report because there are 2 self-citations in the references. Many case reports discuss FB in children, especially in the esophagus.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "10608", "date": "23 Nov 2023", "name": "hamsu kadriyan", "role": "Author Response", "response": "Dear reviewer, Thank you for your comment on my article. Regarding your suggestion, I have revised the article as follows: 1. I have changed the word \"otolaryngologist\" to \"physician\". 2. I have added the anatomy of the esophagus in the discussion section. 3. To prevent self-citation I deleted one of the references, but the other one is significant to support the explanation of health services problem in a rural area. I also added some other references to support the importance of this manuscript." } ] } ]	1	https://f1000research.com/articles/12-7
https://f1000research.com/articles/13-528/v1	22 May 24	{ "type": "Research Article", "title": "Effects of dynamic versus static parameter-guided fluid resuscitation in patients with sepsis: A randomized controlled trial", "authors": [ "Thiti Sricharoenchai", "Pannarat Saisirivechakun", "Pannarat Saisirivechakun" ], "abstract": "Background Fluid resuscitation is an essential component for sepsis treatment. Although several studies demonstrated that dynamic variables were more accurate than static variables for prediction of fluid responsiveness, fluid resuscitation guidance by dynamic variables is not standard for treatment. The objectives were to determine the effects of dynamic inferior vena cava (IVC)-guided versus (vs.) static central venous pressure (CVP)-guided fluid resuscitation in septic patients on mortality; and others, i.e., resuscitation targets, shock duration, fluid and vasopressor amount, invasive respiratory support, length of stay and adverse events.\n\nMethods A single-blind randomized controlled trial was conducted at Thammasat University Hospital between August 2016 and April 2020. Septic patients were stratified by acute physiologic and chronic health evaluation II (APACHE II) <25 or ≥25 and randomized by blocks of 2 and 4 to fluid resuscitation guidance by dynamic IVC or static CVP.\n\nResults Of 124 patients enrolled, 62 were randomized to each group, and one of each was excluded from mortality analysis. Baseline characteristics were comparable. The 30-day mortality rates between dynamic IVC vs. static CVP groups were not different (34.4% vs. 45.9%, p=0.196). Relative risk for 30-day mortality of dynamic IVC group was 0.8 (95%CI=0.5-1.2, p=0.201). Different outcomes were median (interquartile range) of shock duration (0.8 (0.4-1.6) vs. 1.5 (1.1-3.1) days, p=0.001) and norepinephrine (NE) dose (6.8 (3.9–17.8) vs. 16.1 (7.6–53.6) milligrams, p=0.008 and 0.1 (0.1-0.3) vs. 0.3 (0.1-0.8) milligram⋅kilogram−1, p=0.017). Others were not different.\n\nConclusions Dynamic IVC-guided fluid resuscitation does not affect mortality of septic patients. However, this may reduce shock duration and NE dose, compared with static CVP guidance.", "keywords": [ "sepsis", "dynamic parameter", "static parameter", "ultrasound", "fluid resuscitation", "mortality", "norepinephrine", "shock duration" ], "content": "Trial registration\n\nTCTR20160808002 in Thai Clinical Trials Registry (https://www.thaiclinicaltrials.org/export/pdf/TCTR20160808002), Prospectively registered on 3rd August 2016.\n\n\n\n\n\n\n95%CI\n\n95% confidence interval\n\nAPACHE II\n\nacute physiologic and chronic health evaluation II\n\nBMI\n\nbody mass index\n\nBW\n\nbody weight\n\ncIVC\n\ninferior vena cava collapsibility index\n\nCI\n\nconfidence interval\n\ncm\n\ncentimeters\n\nCONSORT\n\nConsolidated Standards of Reporting Trials\n\nCVP\n\ncentral venous pressure\n\ndIVC\n\ninferior vena cava distensibility index\n\nDmax\n\nmaximum diameter\n\nDmin\n\nminimum diameter\n\ne.g.\n\nexempli gratia (for example)\n\net al.\n\net alia (and others)\n\netc.\n\net cetera (and so forth)\n\nETT\n\nendotracheal intubation\n\nF/U\n\nfollow-up\n\nh\n\nhour(s)\n\nICU\n\nintensive care unit\n\ni.e.\n\nid est (that is)\n\nIQR\n\ninterquartile range\n\nIVC\n\ninferior vena cava\n\nkg\n\nkilogram(s)\n\nkg⋅m−2\n\nkilogram(s) per square meter\n\nKUB\n\nkidney-ureter-bladder\n\nL\n\nliter(s)\n\nLOS\n\nlength of stay\n\nMAP\n\nmean arterial pressure\n\nmin\n\nminute(s)\n\nmg\n\nmilligram(s)\n\nmg⋅kg−1\n\nmilligram(s) per kilogram\n\nmL\n\nmilliliter(s)\n\nmL⋅kg−1\n\nmilliliter(s) per kilogram\n\nmL⋅kg−1⋅h−1\n\nmilliliter(s) per kilogram per hour\n\nmmHg\n\nmillimeters of mercury\n\nmmol⋅L−1\n\nmillimole(s) per liter\n\nMV\n\nmechanical ventilation\n\nn\n\nnumber\n\nNaCl\n\nsodium chloride\n\nNE\n\nnorepinephrine\n\np\n\np-value\n\nqSOFA\n\nquick Sequential (Sepsis-Related) Organ Failure Assessment Score\n\nSBP\n\nsystolic blood pressure\n\nScvO2\n\ncentral venous oxygen saturation\n\nSD\n\nstandard deviation\n\nSOFA\n\nSequential (Sepsis-Related) Organ Failure Assessment Score\n\nSSC\n\nSurviving Sepsis Campaign Guidelines\n\nUS\n\nultrasound\n\nvs.\n\nversus\n\n\nIntroduction\n\nSepsis and septic shock potentially cause high mortality and burden for patients and health care system.1,2 Rivers, et al. introduced early goal-directed therapy as the mainstay treatment of sepsis, which has been able to reduce the mortality rate of patients with sepsis or septic shock for more than two decades.3 The Surviving Sepsis Campaign Bundle 2018 Update recommends to initiate rapid administration of intravenous crystalloid solution, at least 30 milliliters per kilogram (mL⋅kg−1), in the first hour for resuscitation in the early phase of sepsis4 because most patients with sepsis are affected by hypovolemic status. Adequate cardiac preload is an essential component for hemodynamic restoration in patients with sepsis. Nevertheless, excessive fluid resuscitation may occur and harm patients with sepsis.5–11 Several physiologic studies have shown that dynamic variables, e.g., pulse pressure variation,12,13 stroke volume variation,14–16 respiratory change in aortic blood velocity,17,18 vena cava diameter variation,19–22 etc. were more accurate for assessment of fluid responsiveness,23 while systematic reviews found that static central venous pressure (CVP) was not highly reliable for assessment of fluid responsiveness.24,25 Surviving Sepsis Campaign Guidelines 2016 suggest using dynamic variables over static variables to guide fluid resuscitation,26 but currently, dynamic variables are not yet standard for prediction of fluid responsiveness.\n\nThis study aimed to determine the effects of dynamic inferior vena cava (IVC)-guided versus static CVP-guided fluid resuscitation in patients with sepsis on mortality and other clinical outcomes, i.e., targets of resuscitation, duration of shock, amount of fluid and vasopressor administration, invasive respiratory support, length of stay (LOS) in hospital and intensive care unit (ICU), and adverse events.\n\n\nMethods\n\nThis was a single-blind randomized controlled trial conducted at the emergency department, general medical wards and medical ICU of Thammasat University Hospital (TUH) between August 2016 and April 2020. The patients were screened if they met all of the following criteria: 1. age of 15 years or more; 2. clinical suspicion of infection with quick Sequential (Sepsis-Related) Organ Failure Assessment Score (qSOFA) of 2 or more27; 3. evidence of organ dysfunction shown by Sequential (Sepsis-Related) Organ Failure Assessment Score (SOFA) of 2 or more27; 4. unstable hemodynamics (systolic blood pressure (SBP) <90 millimeters of mercury (mmHg) or decrease in SBP >40 mmHg from baseline or mean arterial pressure (MAP) <70 mmHg) for 6 hours (h) or less; 5. fluid resuscitation required for unstable hemodynamics. The exclusion criteria were one or more of the following: 1. pregnant women; 2. cardiogenic pulmonary edema; 3. inability to lie down (e.g., scoliosis); 4. limited measurement of IVC diameter by ultrasound (US) (e.g., abdominal mass compressing IVC); 5. difficulty in performing central venous catheterization or CVP measurement (e.g., superior vena cava obstruction). The eligible patients who had given consent were included in the study. PS recruited and stratified patients by acute physiologic and chronic health evaluation II (APACHE II) of <25 or ≥25, then randomized them by blocks of 2 and 4 using sealed envelopeTM program, to either measure IVC diameter variation by US or measure static CVP by central venous catheter and transducer in a ratio of 1:1, in order to guide fluid resuscitation. The allocation sequences in sealed envelopes were disclosed one by one when each patient was randomized. The process of fluid resuscitation guidance was covered from each patient by a screen.\n\nStatic CVP measurement group\n\nThe patients in the static CVP measurement group received central venous catheterization at right or left internal jugular vein by attending physicians. The first CVP measurement was done at the beginning of the study: CVP <8 mmHg and ≥8 mmHg were considered as fluid responsiveness and fluid non-responsiveness, respectively. Fluid-responsive patients were resuscitated by crystalloid solution (mostly 0.9% sodium chloride (NaCl) solution) 500 milliliters (mL) in 15 minutes (min), then CVP was measured repeatedly. Continuous fluid resuscitations and CVP measurements were performed alternately in the same manner until fluid non-responsiveness occurred (i.e., CVP ≥8 mmHg). Then patients with MAP <65 mmHg and fluid non-responsiveness received vasopressor(s) and/or inotropic agent(s) according to Surviving Sepsis Campaign Guidelines 2012,28 intensive monitoring for 6 h, and were followed until shock recovery (i.e., MAP ≥65 mmHg and adequate tissue perfusion without vasopressor(s)), transfer or death within 30 days. Regardless of whether patients recovered from septic shock, they would receive a standard of care from TUH until discharge, transfer or death.\n\nDynamic IVC measurement group\n\nThe patients in the dynamic IVC measurement group were divided into 2 subgroups: mechanically ventilated and spontaneously breathing. The US probe was placed subcostally in order to measure the IVC diameter at 3-4 centimeters (cm) from the IVC-right atrium junction29 as Figure 1. while the patient was in a supine position. The first measurement of respiratory IVC diameter variation by US was done at the beginning of the study in both subgroups. Mechanically ventilated patients were measured for IVC distensibility index ((maximum diameter – minimum diameter)100%/minimum diameter, (Dmax-Dmin)100%/Dmin) with ≥18% and <18% considered as fluid responsiveness, and fluid non-responsiveness, respectively.30 The maximum IVC diameter and minimum IVC diameter occurred during inspiration and expiration, respectively, in mechanically ventilated patients. Spontaneously breathing patients were measured for IVC collapsibility index ((Dmax-Dmin)100%/Dmax) with ≥50% and <50% considered as fluid responsiveness, and fluid non-responsiveness, respectively.31 In contrast, the maximum IVC diameter and minimum IVC diameter occurred during expiration and inspiration, respectively, for spontaneously breathing patients. Respiratory variation of IVC diameter measurement by US was used in this study because of feasibility and availability of US in our hospital settings. The measurement of respiratory variation of IVC diameter using US was performed by PS after training with 30 patients for 2 weeks and supervised by an intensivist. Dynamic IVC diameter measurement was done by using a LOGIQ C5 Premium Ultrasound Machine (GE Healthcare, Chicago, Illinois, USA). Sedative agents (midazolam and/or fentanyl) and neuromuscular blocking agents (atracurium or cisatracurium) were possibly administered in some mechanically ventilated patients to minimize respiratory effort, and maximize the validity of IVC distensibility index. Fluid-responsive patients were resuscitated continuously by crystalloid solution (also mostly 0.9% NaCl solution) 500 mL in 15 min, and IVC distensibility index or IVC collapsibility index measurements were done repeatedly and alternately until fluid non-responsiveness was achieved (i.e., IVC distensibility index <18% or IVC collapsibility index <50%) in the same way as the static CVP measurement group. The vasopressor(s) and inotropic agent(s) administration, including other treatment, monitoring and follow-up processes after the patients meeting fluid non-responsiveness until discharge, transfer or death within 30 days were similar to those of the static CVP measurement group. The flow diagram of study protocol is shown in Figure 2.\n\nAbbreviation: IVC, inferior vena cava.\n\na The photo was taken by TS and was not used or previously published in any journal before.\n\nAbbreviations: APACHE II, acute physiologic and chronic health evaluation II; cIVC, inferior vena cava collapsibility index; CVP, central venous pressure; dIVC, inferior vena cava distensibility index; F/U, follow-up; h, hours; IV, intravenous; IVC, inferior vena cava; L, liter; MAP, mean arterial pressure; min, minutes; mmHg, millimeters of mercury; SSC, Surviving Sepsis Campaign Guidelines.\n\nThe patients’ data were recorded for baseline clinical characteristics (i.e., sex, age, weight, height, comorbidities), characteristics of sepsis (i.e., vital signs, confirmed or suspected source of infection, APACHE II at time of diagnosis), ICU admission, outcomes (i.e., survival status at 30 days after participation in the study, macrovascular targets (MAP, urine output) and microvascular targets (central venous oxygen saturation (ScvO2), serum lactate) at time of diagnosis of sepsis and 6 h later, time of treatment initiation and MAP ≥65 mmHg with adequate tissue perfusion, total volume of fluid administration in first 72 h of sepsis, accumulated dose of norepinephrine (NE), status of endotracheal intubation (ETT) with mechanical ventilation (MV) throughout study period, dates of initiation and termination of MV, dates of hospital admission and discharge, dates of ICU admission and discharge), and adverse events (i.e., fluid overload and pneumothorax in 30 days after participation in the study). The primary outcome was the 30-day mortality of patients; those who were discharged or transferred to other hospitals before 30 days were tracked for survival status at 30 days by telephone. The secondary outcomes were achievements of macrovascular targets (MAP ≥65 mmHg, urine output ≥0.5 milliliter(s) per kilogram per hour (mL⋅kg−1⋅h−1)) and microvascular targets (ScvO2 ≥70%, lactate clearance ≥10%) in the first 6 h,32 duration of shock (time from treatment initiation to MAP ≥65 mmHg and adequate tissue perfusion), total volume of fluid administration and total volume of fluid administration per kilogram (kg) of body weight (BW) in the first 72 h, accumulated dose of NE and accumulated dose of NE per kg of BW, ETT with MV, MV duration, hospital LOS, ICU LOS and adverse events. The clinical outcomes were selected based on feasibility and standard practice of TUH.\n\nThe sample size was estimated to be 122 patients (61 for each group) for the detection of 24% difference in 30-day mortality rate between the two intervention groups33 with an alpha of 0.05 and a power of 80%. Interim analysis of efficacy was performed when the number of patients were recruited up to 50% of sample size. The trial would be terminated if 30-day mortality rates between two intervention groups were extremely different (Haybittle-Peto boundary with an alpha level of 0.002). Baseline clinical characteristics, primary and secondary outcomes were analyzed by descriptive statistics for all patients and each intervention group. Categorical variables were shown as proportion (percentage), and continuous variables were shown as mean ± standard deviation (SD) or median (interquartile range, IQR) as appropriate. Data of each intervention group were compared using Chi-square test for categorical variables, and using Independent two-sample t-test for normally distributed continuous variables or Mann-Whitney U test for non-normally distributed continuous variables. Univariable relative risk regression or univariable Poisson regression analysis was performed for binary outcomes to obtain the relative risk of dynamic IVC measurement-guided fluid resuscitation for each outcome, compared with static CVP measurement-guided fluid resuscitation. The 2-sided p-value (p) <0.05 was considered as statistical significance for comparison between the two groups and regression analysis. All data were analyzed using Stata SE, version 14.0 (StataCorp, College Station, Texas, USA), of which copyright license has been held by the Faculty of Medicine, Thammasat University. An open-access alternative software for statistical analysis is RStudio, which is available from https://posit.co/download/rstudio-desktop/.\n\nThis study was conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving human subjects. Ethics approval for research conduct, documentation of consent from each patient or their representative and information sheet was provided by the Human Research Ethics Committee of Thammasat University (Medicine) (Approval number: 127/2559) on 15th July 2016. A written informed consent for the study was obtained from each patient or their legally authorized representative. No identifiable data of individual persons are presented. Consent for publication was a part of written informed consent for the study, which declared that no identifiable data of individual persons are presented.\n\n\nResults\n\nA total of 995 patients with sepsis or septic shock treated at the emergency department or medical wards in TUH between August 2016 and April 2020 were screened for eligibility, and 124 patients were included. After stratification by APACHE II of <25 or ≥25, 62 patients were randomized into each intervention group. Individual patients were followed until shock recovery, transfer or death within 30 days. One patient in the dynamic IVC measurement group withdrew after randomization because of discomfort for data collection, leaving 123 patients included in the modified intention-to-treat analysis. One additional patient in the static CVP measurement group was excluded from analysis for the 30-day mortality because they were referred to another hospital and their survival status could not be tracked. The study process from enrollment to analysis is shown as a Consolidated Standards of Reporting Trials (CONSORT) flow diagram in Figure 3. The study was ended after completion of patient recruitment and follow-up.\n\nAbbreviations: APACHE II, acute physiologic and chronic health evaluation II; CONSORT, Consolidated Standards of Reporting Trials; CVP, central venous pressure; IVC, inferior vena cava.\n\nOne patient withdrew informed consent because of discomfort for data collection.\n\n*One patient was transferred to other hospital on the same day of enrollment in the study, and their survival status at 30 days could not be tracked.\n\nThe baseline characteristics and severity of sepsis, including ICU admission, were not significantly different between the two intervention groups, as shown in Table 1. Of 111 patients (90.2%) who required NE, the number of patients in the dynamic IVC measurement group (52, 85.3%) was lower than that in the static CVP measurement group (59, 95.2%), but not statistically different (p = 0.064). The primary outcome and most secondary outcomes were not significantly different between both groups, i.e., mortality rates in 30 days, achievement of macrovascular target(s) (MAP ≥65 mmHg or urine output ≥0.5 mL⋅kg−1⋅h−1 or both) in 6 h or achievement of microvascular target(s) (ScvO2 ≥70% or lactate clearance ≥10% or both) in 6 h, as well as total volume of fluid and total volume of fluid per kg of BW administered in the first 72 h of sepsis, ETT with MV, duration of MV, hospital LOS and ICU LOS. However, the dynamic IVC measurement group demonstrated significantly shorter median (IQR) duration of shock, considerably lower median (IQR) accumulated dose of NE and lower median (IQR) accumulated dose of NE per kg of BW. Rates of adverse events were not different between the two intervention groups. All outcomes in detail are shown in Table 2. Thirty-day mortality rates and significantly different secondary outcomes (i.e., duration of shock, accumulated dose of NE and accumulated dose of NE per kg of BW) are presented in Extended data Figure S1., S2., S3. and S4. All findings were based on analyses of the dataset from this study.34\n\na This characteristic was analyzed from 101 patients (39 in Dynamic IVC measurement group, and 62 in Static CVP measurement group) who received central venous catheterization.\n\n$ Chi-square test.\n\n@ Independent two-sample t-test.\n\n# Mann-Whitney U test.\n\nα Relative risk regression.\n\nβ Poisson regression.\n\n Statistically significant different.\n\na This outcome was analyzed from 122 patients (61 in Dynamic IVC measurement group, and 61 in Static CVP measurement group) because one patient in static CVP group was transferred to other hospital and their survival status at 30 days could not be tracked.\n\nb This outcome was analyzed from 101 patients who received central venous catheterization (39 in Dynamic IVC measurement group, and 62 in Static CVP measurement group).\n\nc This outcome was analyzed from 114 patients who can be assessed for achievement of ≥1 microvascular target in 6 hours (52 in Dynamic IVC measurement group, and 62 in Static CVP measurement group).\n\nd This outcome was analyzed from 110 patients who can be assessed for achievement of 2 microvascular targets in 6 hours (48 in Dynamic IVC measurement group, and 62 in Static CVP measurement group).\n\ne These outcomes were analyzed from 73 survived patients (40 in Dynamic IVC measurement group, and 33 in Static CVP measurement group).\n\nf These outcomes were analyzed from 65 survived patients who required norepinephrine (33 in Dynamic IVC measurement group, and 32 in Static CVP measurement group).\n\ng This outcome was analyzed from 32 survived patients who required endotracheal intubation and mechanical ventilation (15 in Dynamic IVC measurement group, and 17 in Static CVP measurement group).\n\nh This outcome was analyzed from 22 survived patients who were admitted to ICU (12 in Dynamic IVC measurement group, and 10 in Static CVP measurement group).\n\n\nDiscussion\n\nOur study showed that the baseline characteristics were not different between the two intervention groups. The 30-day mortality rates were not different between both groups, nor were achievement of macrovascular and microvascular targets in 6 h, total volume of fluid administration and total volume of fluid administration per kg of BW in the first 72 h of sepsis, rate of ETT with MV, duration of MV, hospital LOS, ICU LOS and rates of adverse events. However, patients in the dynamic IVC measurement group experienced shorter duration of shock, lower accumulated dose of NE and lower accumulated dose of NE per kg of BW, compared with those in the static CVP measurement group.\n\nTo our knowledge, this is the first study attempting to determine the clinical effects of fluid resuscitation guided by feasible and practical hemodynamic parameter for prediction of fluid responsiveness in patients with sepsis or septic shock in Thailand. There was no difference in the 30-day mortality rates between the two groups, which was consistent with the 28-day mortality rate of septic shock patients in a study from Richard, et al.33 Although the 30-day mortality rates in the two groups were not statistically different, the percentage of mortality in the dynamic parameter group was somewhat lower (absolute difference about 10%) than in the static parameter group in our study, and was even considerably lower (absolute difference about 20%) in that study.33 This may imply important clinical significance in certain clinical situations, especially for the disease with high prevalence like sepsis, because the absolute number of deaths would be magnified by the prevalence of the disease. The mortality rates (at 30 days,35,36 90 days,37 180 days,35 and hospital mortality38) of surgical ICU patients between fluid therapy guided by dynamic variables and standard care (fluid therapy guided by static variables or clinical examination) were also not different in other studies. However, a recent systematic review and meta-analysis including studies of critically ill adult patients requiring acute fluid resuscitation demonstrated that dynamic variable-guided fluid therapy decreased the mortality risk (risk ratio = 0.59, 95%CI = 0.42-0.83, p = 0.002) compared with standard care.39 The difference between our findings and the systematic review findings in terms of mortality may be partially explained by the different types of patients and therefore different pathophysiologies of diseases. The majority of patients in our study were medical patients while most patients in that systematic review were surgical patients. Medical patients usually have more comorbidities, which poses a higher risk of complications and/or death, compared with surgical patients, and this factor may interfere with the effect of intervention on mortality.\n\nShorter duration of shock and lower accumulated dose of NE (either total dose or total dose per kg of BW) in the dynamic IVC measurement group in our study suggests that dynamic IVC diameter variation may be able to predict fluid responsiveness better than static CVP value, so the patients received more adequate volume of fluid administration. This assumption corresponds to the study findings from Lanspa, et al., which showed that IVC collapsibility index threshold of ≥50% had a fair positive predictive value (75%) and a good negative predictive value (80%) for prediction of fluid responsiveness, although this did not reach statistical significance (p = 0.09).31 Theoretically, patients with adequate fluid volume administration can likely maintain macrovascular and microvascular targets, and minimize duration of shock without excessive dose of vasopressors, resulting in fewer complications from prolonged shock or high dose of vasopressors.40–42 However, patients in the dynamic IVC measurement group did not achieve more macrovascular or microvascular target(s) in 6 h than those in the other group in our study. The reason for this may be the fact that the timing of vasopressor initiation was not controlled because it was decided based on the individual attending physician’s discretion. Given that a higher proportion of patients who received vasopressor earlier could possibly restore macrovascular and microvascular targets as demonstrated in a study from Permpikul, et al. (CENSER trial),43 this may confound the effect of fluid administration guidance method on achievement of macrovascular and microvascular targets.\n\nIn our study, total fluid volume and total fluid volume per kg of BW administered in the first 72 h were not different between the two intervention groups, which was consistent with a study from Trof, et al.44 but different from a study by Richard, et al. which showed lower daily intravenous fluid volume administration in a dynamic variable-guided fluid therapy group.33 The reason for different findings can be partially explained by different time-frames of fluid administration and also different dynamic parameters guiding fluid resuscitation between our study and that study.33 Moreover, our study protocol did not control fluid administration after shock recovery occurring within 72 h, which affected total fluid volume and total fluid volume per kg of BW in the first 72 h. Another study by Douglas, et al. demonstrated less positive fluid balance at 72 h or ICU discharge in septic patients who received dynamic stroke volume-guided fluid resuscitation, compared to those with standard usual care,45 but this outcome cannot be directly compared to total fluid volume in 72 h of our study.\n\nOur study found no difference in rates of ETT and MV between the two intervention groups. This was a reasonable finding because indications of intubation and MV are broader than only unstable hemodynamics. Durations of MV were also not different between the two intervention groups in our study, which was the same as studies of Richard, et al.,33 Douglas, et al.45 and Trof, et al., specifically for septic shock patients.44\n\nHospital LOS in both intervention groups were not different, which was similar to some studies35,45–47 but different from others (shorter hospital LOS in dynamic variable-guided fluid therapy group).36,48–52 ICU LOS were not different between our two intervention groups, which also was consistent with several studies33,37,45,46,49 but different from others.47,51,52 The heterogeneous findings in hospital LOS and ICU LOS were likely caused by the different nature of diseases, given that most patients in these studies were various types of surgical patients, and there were specific indications for ICU admission for certain types of surgical patients (e.g., postoperative observation).\n\nThere are some limitations in this study. Firstly, the sample size was quite small and possibly underpowered to be able to detect the differences of some secondary outcomes between the two intervention groups. A study with a larger sample size is warranted to clarify whether such outcomes are different. However, our sample size was comparable or even greater than other studies assessing the effects on clinical outcomes between dynamic and static hemodynamic parameters.33,44,53 Second, treatment of sepsis for each patient was based on individual attending physicians, except for fluid resuscitation and intensive monitoring in the early phase of sepsis. This might have interfered with the effects of intervention, but clinical practices were standardized by following the Surviving Sepsis Campaign Guidelines4,26,28 and standard of care at TUH under supervision of specialists. Third, the US measurement process of respiratory variation of IVC diameter is operator-dependent. Accordingly, all US measurements of IVC diameter variation were done by the same well-trained physician and the same US machine to eradicate interobserver variability and standardize the findings of US measurement. Fourth, the parameters for prediction of fluid responsiveness in our study were derived from right-sided cardiac preload, which indirectly reflects the variation of stroke volume or cardiac output of the left ventricle after fluid administration. Nevertheless, right-sided cardiac preload-derived parameters were selected based on availability and practicability. Fifth, this was a single-center study, which may not be able to perfectly generalize the findings to other institutes or other hospitals. Physicians need to carefully consider the context of individual situations before adoption of our study findings for clinical practices. Strengths of this study include study design (randomized controlled trial), which could balance baseline characteristics and severity of sepsis in patients between the two intervention groups in order to minimize confounding factors affecting the outcomes, and feasibility for limited-resource hospitals.\n\n\nConclusions\n\nDynamic IVC measurement-guided fluid resuscitation in patients with sepsis or septic shock does not affect mortality, macrovascular and microvascular targets, volume of fluid administration, ETT with MV, duration of MV, hospital LOS, ICU LOS and adverse events. Nevertheless, such intervention may decrease the duration of shock and accumulated dose of NE, compared with static CVP measurement-guided fluid resuscitation.\n\n\nAuthor contributions\n\nConceptualization: Sricharoenchai T. and Saisirivechakun P.; Data curation: Sricharoenchai T. and Saisirivechakun P.; Formal analysis: Sricharoenchai T. and Saisirivechakun P.; Investigation: Sricharoenchai T. and Saisirivechakun P.; Methodology: Sricharoenchai T. and Saisirivechakun P.; Project administration: Sricharoenchai T. and Saisirivechakun P.; Resources: Sricharoenchai T. and Saisirivechakun P.; Software: Sricharoenchai T. and Saisirivechakun P.; Supervision: Sricharoenchai T.; Validation: Sricharoenchai T.; Visualization: Sricharoenchai T. and Saisirivechakun P.; Writing − Original Draft Preparation: Sricharoenchai T.; Writing − Review & Editing: Sricharoenchai T. and Saisirivechakun P.", "appendix": "Data availability statement\n\nZenodo: Data for effects of dynamic vs. static parameter-guided fluid resuscitation in patients with sepsis, https://zenodo.org/records/10579408 or DOI: 10.5281/zenodo.10579407. 34\n\nThis project contains the following underlying data:\n\n• Data for dynamic vs. static parameter-guided resuscitation in sepsis.dta (baseline characteristics and outcomes of patients with sepsis)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo: Supplementary materials for effects of dynamic vs. static parameter-guided fluid resuscitation in patients with sepsis, https://zenodo.org/records/10594154 (Sricharoenchai 2024) or DOI: 10.5281/zenodo.10594153.\n\nThis project contains the following extended data:\n\n• Figure S1. Bar graphs showing the 30-day mortality rate. (30-day mortality rate between dynamic IVC measurement and static CVP measurement groups)\n\n• Figure S2. Box and whisker plots showing the median (IQR) duration of shock. (median (IQR) duration of shock between dynamic IVC measurement and static CVP measurement groups)\n\n• Figure S3. Box and whisker plots showing the median (IQR) accumulated dose of NE. (median (IQR) accumulated dose of NE between dynamic IVC measurement and static CVP measurement groups)\n\n• Figure S4. Box and whisker plots showing the median (IQR) accumulated dose of NE per kg of body weight. (median (IQR) accumulated dose of NE per kg of body weight between dynamic IVC measurement and static CVP measurement groups)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo: A completed CONSORT 2010 checklist for Effects of dynamic versus static parameter-guided fluid resuscitation in patients with sepsis: A randomized controlled trial, https://zenodo.org/records/10597483 (Sricharoenchai 2024) or DOI: 10.5281/zenodo.10597239.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors would like to thank Nanthawan Okas (Department of Medicine, Faculty of Medicine, Thammasat University) for assistance with data collection, Assoc. Prof. Pattarin Pirompanich (Division of Pulmonary and Critical Care Medicine, Department of Medicine, Faculty of Medicine, Thammasat University) for training and supervising Pannarat Saisirivechakun to measure respiratory variation of IVC diameter by US, and Sam Ormond (Faculty of Medicine, Thammasat University) for improving the English language of this manuscript. Permission from each of those people in our acknowledgments has been granted.\n\n\nReferences\n\nAngus DC, Linde-Zwirble WT, Lidicker J, et al.: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 2001; 29: 1303–1310. Publisher Full Text\n\nMartin GS, Mannino DM, Eaton S, et al.: The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 2003; 348: 1546–1554. PubMed Abstract \| Publisher Full Text\n\nRivers E, Nguyen B, Havstad S, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N. Engl. J. Med. 2001; 345: 1368–1377. Publisher Full Text\n\nLevy MM, Evans LE, Rhodes A: The Surviving Sepsis Campaign Bundle: 2018 update. Intensive Care Med. 2018; 44: 925–928. PubMed Abstract \| Publisher Full Text\n\nMarik PE, Linde-Zwirble WT, Bittner EA, et al.: Fluid administration in severe sepsis and septic shock, patterns and outcomes: an analysis of a large national database. Intensive Care Med. 2017; 43: 625–632. PubMed Abstract \| Publisher Full Text\n\nAcheampong A, Vincent JL: A positive fluid balance is an independent prognostic factor in patients with sepsis. Crit. Care. 2015; 19: 251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrotfain E, Koyfman L, Toledano R, et al.: Positive fluid balance as a major predictor of clinical outcome of patients with sepsis/septic shock after ICU discharge. Am. J. Emerg. Med. 2016; 34: 2122–2126. PubMed Abstract \| Publisher Full Text\n\nMitchell KH, Carlbom D, Caldwell E, et al.: Volume Overload: Prevalence, Risk Factors, and Functional Outcome in Survivors of Septic Shock. Ann. Am. Thorac. Soc. 2015; 12: 1837–1844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Oliveira FS, Freitas FG, Ferreira EM, et al.: Positive fluid balance as a prognostic factor for mortality and acute kidney injury in severe sepsis and septic shock. J. Crit. Care. 2015; 30: 97–101. PubMed Abstract \| Publisher Full Text\n\nVincent JL, Sakr Y, Sprung CL, et al.: Sepsis in European intensive care units: results of the SOAP study. Crit. Care Med. 2006; 34: 344–353. PubMed Abstract \| Publisher Full Text\n\nBoyd JH, Forbes J, Nakada TA, et al.: Fluid resuscitation in septic shock: a positive fluid balance and elevated central venous pressure are associated with increased mortality. Crit. Care Med. 2011; 39: 259–265. PubMed Abstract \| Publisher Full Text\n\nDe Backer D, Heenen S, Piagnerelli M, et al.: Pulse pressure variations to predict fluid responsiveness: influence of tidal volume. Intensive Care Med. 2005; 31: 517–523. PubMed Abstract \| Publisher Full Text\n\nMichard F, Boussat S, Chemla D, et al.: Relation between respiratory changes in arterial pulse pressure and fluid responsiveness in septic patients with acute circulatory failure. Am. J. Respir. Crit. Care Med. 2000; 162: 134–138. PubMed Abstract \| Publisher Full Text\n\nCannesson M, Musard H, Desebbe O, et al.: The ability of stroke volume variations obtained with Vigileo/FloTrac system to monitor fluid responsiveness in mechanically ventilated patients. Anesth. Analg. 2009; 108: 513–517. PubMed Abstract \| Publisher Full Text\n\nMarx G, Cope T, McCrossan L, et al.: Assessing fluid responsiveness by stroke volume variation in mechanically ventilated patients with severe sepsis. Eur. J. Anaesthesiol. 2004; 21: 132–138. Publisher Full Text\n\nHofer CK, Senn A, Weibel L, et al.: Assessment of stroke volume variation for prediction of fluid responsiveness using the modified FloTrac and PiCCOplus system. Crit. Care. 2008; 12: R82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonnet X, Rienzo M, Osman D, et al.: Esophageal Doppler monitoring predicts fluid responsiveness in critically ill ventilated patients. Intensive Care Med. 2005; 31: 1195–1201. PubMed Abstract \| Publisher Full Text\n\nFeissel M, Michard F, Mangin I, et al.: Respiratory changes in aortic blood velocity as an indicator of fluid responsiveness in ventilated patients with septic shock. Chest. 2001; 119: 867–873. PubMed Abstract \| Publisher Full Text\n\nFeissel M, Michard F, Faller JP, et al.: The respiratory variation in inferior vena cava diameter as a guide to fluid therapy. Intensive Care Med. 2004; 30: 1834–1837. PubMed Abstract \| Publisher Full Text\n\nVieillard-Baron A, Chergui K, Rabiller A, et al.: Superior vena caval collapsibility as a gauge of volume status in ventilated septic patients. Intensive Care Med. 2004; 30: 1734–1739. PubMed Abstract \| Publisher Full Text\n\nMoretti R, Pizzi B: Inferior vena cava distensibility as a predictor of fluid responsiveness in patients with subarachnoid hemorrhage. Neurocrit. Care. 2010; 13: 3–9. PubMed Abstract \| Publisher Full Text\n\nWang Y, Jiang Y, Wu H, et al.: Assessment of fluid responsiveness by inferior vena cava diameter variation in post-pneumonectomy patients. Echocardiography. 2018; 35: 1922–1925. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichard F, Teboul JL: Predicting fluid responsiveness in ICU patients: a critical analysis of the evidence. Chest. 2002; 121: 2000–2008. Publisher Full Text\n\nMarik PE, Cavallazzi R: Does the central venous pressure predict fluid responsiveness? An updated meta-analysis and a plea for some common sense. Crit. Care Med. 2013; 41: 1774–1781. PubMed Abstract \| Publisher Full Text\n\nEskesen TG, Wetterslev M, Perner A: Systematic review including re-analyses of 1148 individual data sets of central venous pressure as a predictor of fluid responsiveness. Intensive Care Med. 2016; 42: 324–332. PubMed Abstract \| Publisher Full Text\n\nRhodes A, Evans LE, Alhazzani W, et al.: Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016. Intensive Care Med. 2017; 43: 304–377. PubMed Abstract \| Publisher Full Text\n\nSinger M, Deutschman CS, Seymour CW, et al.: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016; 315: 801–810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDellinger RP, Levy MM, Rhodes A, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med. 2013; 39: 165–228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPreau S, Bortolotti P, Colling D, et al.: Diagnostic Accuracy of the Inferior Vena Cava Collapsibility to Predict Fluid Responsiveness in Spontaneously Breathing Patients With Sepsis and Acute Circulatory Failure. Crit. Care Med. 2017; 45: e290–e297. PubMed Abstract \| Publisher Full Text\n\nBarbier C, Loubieres Y, Schmit C, et al.: Respiratory changes in inferior vena cava diameter are helpful in predicting fluid responsiveness in ventilated septic patients. Intensive Care Med. 2004; 30: 1740–1746. PubMed Abstract \| Publisher Full Text\n\nLanspa MJ, Grissom CK, Hirshberg EL, et al.: Applying dynamic parameters to predict hemodynamic response to volume expansion in spontaneously breathing patients with septic shock. Shock. 2013; 39: 155–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArnold RC, Shapiro NI, Jones AE, et al.: Multicenter study of early lactate clearance as a determinant of survival in patients with presumed sepsis. Shock. 2009; 32: 35–39. PubMed Abstract \| Publisher Full Text\n\nRichard JC, Bayle F, Bourdin G, et al.: Preload dependence indices to titrate volume expansion during septic shock: a randomized controlled trial. Crit. Care. 2015; 19: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSricharoenchai T, Saisirivechakun P: Data for effects of dynamic vs. static parameter-guided fluid resuscitation in patients with sepsis. Pathum Thani, Thailand: Thammasat University; 2024. [cited 31 Jan 2024]. Reference Source\n\nPearse RM, Harrison DA, MacDonald N, et al.: Effect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review. JAMA. 2014; 311: 2181–2190. PubMed Abstract \| Publisher Full Text\n\nColantonio L, Claroni C, Fabrizi L, et al.: A randomized trial of goal directed vs. standard fluid therapy in cytoreductive surgery with hyperthermic intraperitoneal chemotherapy. J. Gastrointest. Surg. 2015; 19: 722–729. PubMed Abstract \| Publisher Full Text\n\nParke RL, McGuinness SP, Gilder E, et al.: A randomised feasibility study to assess a novel strategy to rationalise fluid in patients after cardiac surgery. Br. J. Anaesth. 2015; 115: 45–52. PubMed Abstract \| Publisher Full Text\n\nJhanji S, Vivian-Smith A, Lucena-Amaro S, et al.: Haemodynamic optimisation improves tissue microvascular flow and oxygenation after major surgery: a randomised controlled trial. Crit. Care. 2010; 14: R151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBednarczyk JM, Fridfinnson JA, Kumar A, et al.: Incorporating Dynamic Assessment of Fluid Responsiveness Into Goal-Directed Therapy: A Systematic Review and Meta-Analysis. Crit. Care Med. 2017; 45: 1538–1545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalis JU, Rappaport ES, Gerber L, et al.: A primate model for prolonged endotoxin shock. Blood-vascular reactions and effects of glucocorticoid treatment. Lab. Investig. 1978; 38: 511–523. PubMed Abstract\n\nHollenberg SM: Vasoactive drugs in circulatory shock. Am. J. Respir. Crit. Care Med. 2011; 183: 847–855. Publisher Full Text\n\nAuchet T, Regnier MA, Girerd N, et al.: Outcome of patients with septic shock and high-dose vasopressor therapy. Ann. Intensive Care. 2017; 7: 43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPermpikul C, Tongyoo S, Viarasilpa T, et al.: Early Use of Norepinephrine in Septic Shock Resuscitation (CENSER). A Randomized Trial. Am. J. Respir. Crit. Care Med. 2019; 199: 1097–1105. PubMed Abstract \| Publisher Full Text\n\nTrof RJ, Beishuizen A, Cornet AD, et al.: Volume-limited versus pressure-limited hemodynamic management in septic and nonseptic shock. Crit. Care Med. 2012; 40: 1177–1185. PubMed Abstract \| Publisher Full Text\n\nDouglas IS, Alapat PM, Corl KA, et al.: Fluid Response Evaluation in Sepsis Hypotension and Shock: A Randomized Clinical Trial. Chest. 2020; 158: 1431–1445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuettner M, Schummer W, Huettemann E, et al.: Influence of systolic-pressure-variation-guided intraoperative fluid management on organ function and oxygen transport. Br. J. Anaesth. 2008; 101: 194–199. PubMed Abstract \| Publisher Full Text\n\nKumar L, Rajan S, Baalachandran R: Outcomes associated with stroke volume variation versus central venous pressure guided fluid replacements during major abdominal surgery. J. Anaesthesiol. Clin. Pharmacol. 2016; 32: 182–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopes MR, Oliveira MA, Pereira VO, et al.: Goal-directed fluid management based on pulse pressure variation monitoring during high-risk surgery: a pilot randomized controlled trial. Crit. Care. 2007; 11: R100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayer J, Boldt J, Mengistu AM, et al.: Goal-directed intraoperative therapy based on autocalibrated arterial pressure waveform analysis reduces hospital stay in high-risk surgical patients: a randomized, controlled trial. Crit. Care. 2010; 14: R18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng H, Guo H, Ye JR, et al.: Goal-directed fluid therapy in gastrointestinal surgery in older coronary heart disease patients: randomized trial. World J. Surg. 2013; 37: 2820–2829. PubMed Abstract \| Publisher Full Text\n\nGoepfert MS, Richter HP, Zu Eulenburg C, et al.: Individually optimized hemodynamic therapy reduces complications and length of stay in the intensive care unit: a prospective, randomized controlled trial. Anesthesiology. 2013; 119: 824–836. PubMed Abstract \| Publisher Full Text\n\nKapoor PM, Kakani M, Chowdhury U, et al.: Early goal-directed therapy in moderate to high-risk cardiac surgery patients. Ann. Card. Anaesth. 2008; 11: 27–34. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Lu YH, Xie JF, et al.: Passive leg raising predicts volume responsiveness in patients with septic shock. Zhonghua wai ke za zhi [Chinese Journal of Surgery]. 2011; 49: 44–48. PubMed Abstract" }	[ { "id": "282204", "date": "14 Jun 2024", "name": "Jhuma Sankar", "expertise": [ "Reviewer Expertise Septic shock", "sepsis", "hemodynamic monitoring", "scoring systems", "anti microbial 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\nAbstract: No comments Introduction: Optimal Methods: The effect size for calculation of sample size is too large 24%. How was this number chosen? An ultrasound method to guide resuscitation may not be able to produce such a large difference in mortality. Ten % at the most would have been optimal. This should be addressed as a limitation. Results: No comments Discussion: Please address the limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "12057", "date": "23 Jul 2024", "name": "Thiti Sricharoenchai", "role": "Author Response", "response": "Reviewer's Comment: The effect size for calculation of sample size is too large 24%. How was this number chosen? An ultrasound method to guide resuscitation may not be able to produce such a large difference in mortality. Ten % at the most would have been optimal. This should be addressed as a limitation. Authors' Response: Thank you so much for your comment. The difference in mortality rate was selected based on the study findings from Richard, et al. (28-day mortality rates in preload dependence group vs. control group were 23% vs. 47%, respectively) (reference 33 of the manuscript).1 However, we agreed that the difference in mortality rate of 24% was quite high. This issue was explained in the limitations of Discussion section. References 1. Richard JC, Bayle F, Bourdin G, et al.: Preload dependence indices to titrate volume expansion during septic shock: a randomized controlled trial. Crit. Care. 2015; 19: 5." } ] }, { "id": "282206", "date": "20 Jun 2024", "name": "Surat Tongyoo", "expertise": [ "Reviewer Expertise Septic shock" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 support this article for approval in F1000Research, but after a minor revision. The following are my comments. 1.Please explain why the author used CVP at 8 mmHg as a cut point for fluid responsiveness. For me it seem to be too low level for that. 2.Conventionally, we used follow up CVP with an elevation of CVP from baseline > 5-7 mmHg as a cut point for fluid responsive, why the author did not use this. 3.For IVC US, author provide 2 cut point of IVC variation for fluid responsive, why you not provide different cut point for CVP as the positive intra-thoracic pressure might increase baseline CVP as well. 4. Please provide more detail in your results manuscript section. 5. Norepinephrine should be microgram/kg/min, not mg. 6. Please discuss more about even patient in both group received fluid in same volume, why they received NE in different dose, and also the different in shock duration.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "12058", "date": "25 Jul 2024", "name": "Thiti Sricharoenchai", "role": "Author Response", "response": "Reviewer's Comment 1. Please explain why the author used CVP at 8 mmHg as a cut point for fluid responsiveness. For me it seem to be too low level for that. Authors' Response: Thank you for your comment. The Surviving sepsis campaign guidelines 2012 (reference 28 of the manuscript)1 recommended to keep CVP at least 8 mmHg to be adequate volume status. The CVP of 8 mmHg was used as the cut-off point for fluid responsiveness in other studies (reference 33 and 51 of the manuscript).2, 3 This CVP cut-off point (8 mmHg) is also usually used in our hospital settings, most attending physicians are familiar with this CVP value for assessment of fluid responsiveness. This would help the process of study to be consistent for all patients in the static CVP group. The explanations were presented in Intervention of Methods section. Reviewer's Comment 2. Conventionally, we used follow up CVP with an elevation of CVP from baseline > 5-7 mmHg as a cut point for fluid responsive, why the author did not use this. Authors' Response: We appreciate your comment. While we agree that an elevation of CVP from baseline > 5-7 mmHg is usually considered as a cut-off point for fluid responsiveness, the elevation of CVP from baseline usually refers to the increased value of CVP after the patient receives fluid administration, which is more likely to be a dynamic parameter, not a static parameter as we intended to determine the effect of fluid resuscitation guidance. Therefore, we used the CVP of 8 mmHg as a cut-off point for assessment of fluid responsiveness. Reviewer's Comment 3. For IVC US, author provide 2 cut point of IVC variation for fluid responsive, why you not provide different cut point for CVP as the positive intra-thoracic pressure might increase baseline CVP as well. Authors' Response: We are grateful for your comment and concern. The Surviving sepsis campaign guidelines 2012 (reference 28 of the manuscript)1 recommended to keep CVP of 8-12 mmHg regardless of respiratory variation of intrathoracic pressure. Our study followed this by considering the CVP of ≥ 8 mmHg as adequate volume status in the static CVP group, as well as the study by Richard, et al. (reference 33 of the manuscript).2 The reasons were provided in Intervention of Methods section. Reviewer's Comment 4. Please provide more detail in your results manuscript section. Authors' Response: We acknowledge your advice. We have revised your concerned issues in Methods, Results and Discussion sections. Reviewer's Comment 5. Norepinephrine should be microgram/kg/min, not mg. Authors' Response: Thank you for your suggestion. We used the rate of norepinephrine as “microgram/kg/min”. However, we calculated further for the dose of norepinephrine to be “mg” as follows: 1) Accumulated dose of norepinephrine received by each patient throughout the total vasopressor duration; the rate of norepinephrine (microgram/kg/min) multiplied by the body weight (kg) and the duration of norepinephrine administration (min), then divided by 1,000. Thus, the accumulated dose of norepinephrine was converted to “mg”. 2) Accumulated dose of norepinephrine per kg of body weight received by each patient throughout the total vasopressor duration; the rate of norepinephrine (microgram/kg/min) was multiplied by the duration of norepinephrine administration (min), then divided by 1,000. Thus, the accumulated dose of norepinephrine was converted to “mg per kg”. The details of unit conversion of norepinephrine were shown in Data collection and outcome measures of Methods section. Reviewer's Comment 6. Please discuss more about even patient in both group received fluid in same volume, why they received NE in different dose, and also the different in shock duration. Authors' Response: We are very grateful for your comment. The total fluid volume administered in the first 72 hours in most patients included the volume of fluid during the shock period and after the resolution of shock until 72 hours, because durations of shock were less than 72 hours in majorities of both groups. Furthermore, fluid volume after the resolution of shock until 72 hours for individual patients depended on the attending physicians’ discretion and was not controlled in this study. Therefore, total fluid volume in the first 72 hours cannot reflect the efficacy of guidance methods for fluid resuscitation, and may not be related to the accumulated dose of norepinephrine and duration of shock. This was explained in Results and Discussion sections. References 1. Dellinger RP, Levy MM, Rhodes A, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med. 2013;39:165–228. 2. Richard JC, Bayle F, Bourdin G, et al.: Preload dependence indices to titrate volume expansion during septic shock: a randomized controlled trial. Crit. Care. 2015;19:5. 3. Goepfert MS, Richter HP, Zu Eulenburg C, et al.: Individually optimized hemodynamic therapy reduces complications and length of stay in the intensive care unit: a prospective, randomized controlled trial. Anesthesiology. 2013;119:824–836." } ] } ]	1	https://f1000research.com/articles/13-528
https://f1000research.com/articles/13-830/v1	24 Jul 24	{ "type": "Brief Report", "title": "Induction of torpor in response to a common chronic food restriction paradigm: implications for behavioural research using mice", "authors": [ "Sian L. Wilcox", "David M. Bannerman", "Stuart N. Peirson", "Vladyslav V. Vyazovskiy", "David M. Bannerman", "Stuart N. Peirson", "Vladyslav V. Vyazovskiy" ], "abstract": "Background Many behavioural, pharmacological, and metabolic studies in mice require fasting, yet the possibility of fasting-induced torpor affecting data is rarely considered. Torpor is a state characterised by depressed metabolism and profound alterations in physiology and behaviour. In this study, we aimed to determine whether a chronic food restriction paradigm, common in behavioural studies, was sufficient to induce torpor in mice.\n\nMethods Mice were food restricted to ~85-90% of their bodyweight, as is typically done, and monitored using continuous thermal imaging.\n\nResults We observed that body temperature significantly decreased over days of food restriction, and it was significantly related to the drop in bodyweight (r2=0.8989, p<0.0001). All mice reliably entered torpor daily from day 8 of food restriction which coincided with bodyweight stabilisation at ~85%. We found a strong positive relationship between the magnitude of the decrease of bodyweight and the proportion of mice entering torpor each day (r2=0.8715, p<0.0001).\n\nConclusions Overall, we found that torpor is readily induced in response to food restriction. Considering that hunger is frequently used as a motivational drive in behavioural tasks, it is likely that torpor occurrence is common in such studies, while remaining undetected and unaccounted for. Due to the profound effect of torpor on physiology, it is possible that torpor induction may be confounding subsequent data and represents an important source of variation. We recommend that body temperature is always monitored noninvasively in studies where food restriction is employed, to determine when torpor is occurring, and that torpor history is appropriately controlled for within and across experimental groups.", "keywords": [ "3Rs", "Daily torpor", "Food restriction", "Refinement", "Mice" ], "content": "\n\n\n\nScientific benefit:\n\n\n\n• Identification of torpor induction in response to food restriction in laboratory mice\n\n• Raising awareness of torpor induction as a result of food restriction (an unwanted side effect which may impact study outcome measures)\n\n3Rs benefit:\n\n\n\n• Identifying and controlling for potential sources of variation in experimental variables due to torpor occurrence\n\n• Welfare implications of food restriction in behavioural studies - inducing torpor as an unwanted side effect\n\n• Thermal imaging, providing a non-invasive measuring of torpor\n\nCurrent applications:\n\n\n\n• Research using food restriction techniques in mice, particularly behavioural neuroscience\n\nPotential applications:\n\n\n\n• Research fields which use food restriction or fasting in mice e.g., metabolic studies, pharmacological studies, behavioural studies\n\n\nIntroduction\n\nThe laboratory mouse is a widely used model organism for scientific research, accounting for 54% (934,200) of experimental procedures performed on animals in the UK in 2021 (Annual Statistics of Scientific Procedures on Living Animals Great Britain 2021). As part of many experimental procedures, fasting and food restriction are frequently used, for example in behavioural and sensory neuroscience (Padamsey et al., 2021; Pioli et al., 2014), metabolic studies (Ayala et al., 2010; Berglund et al., 2008), pharmacological studies (Chen et al., 2018; Huang et al., 2015), and circadian phenotyping (Greenwell et al., 2019; Northeast et al., 2019), amongst others.\n\nThe potential for fasting or food restriction to be a confound for experimental data is rarely considered within studies that utilise this technique, despite fasting being known to significantly alter physiology (Jensen et al., 2013). Food restriction is especially prevalent in behavioural neuroscience as a method for incentivising engagement with a task. Frequently, mice undergo chronic food restriction in which bodyweight is maintained at ~85-90%. However, behavioural data is subject to high levels of inter- and intra-individual variation, therefore requiring large sample sizes, although reproducibility is often limited (Baker & Penny, 2016; Begley & Ioannidis, 2015; Mandillo et al., 2008).\n\nWe hypothesised that torpor, a naturally occurring state of metabolic suppression and hypothermia in response to limited food availability, may be an unaddressed confounding factor in behavioural studies. It has been reported that laboratory mice enter torpor in response to fasting (Ambler et al., 2021; Hudson & Scott, 1979; Jensen et al., 2013), resulting in profound changes to central and peripheral physiology. For example, torpor has been associated with sleep disruption and altered brain activity (Huang et al., 2021; Northeast et al., 2019; Vyazovskiy et al., 2017), and altered levels of circulating hormones such as thyroid hormones (Bank et al., 2015), leptin (Gavrilova et al., 1999), and ghrelin (Gluck et al., 2006).\n\nHere, we aimed to determine the likelihood of torpor induction in response to a food restriction paradigm common in behavioural studies. We focused on the influence of bodyweight due to previous studies demonstrating a link between bodyweight and torpor (Kato et al., 2018; Solymár et al., 2015), and due to weight loss being a key component of food restriction paradigms.\n\n\nMethods\n\nProcedures were performed in compliance with the United Kingdom Animals (Scientific Procedures) Act of 1986 (Project Licence Number: P828B64BC), and the University of Oxford Policy on the Use of Animals in Scientific Research. All experiments performed under this project licence were approved by the University of Oxford Animal Welfare and Ethical Review Board (AWERB) on 28th February 2017 (reference: DPAGEP (17) 9, Item 5a). A retrospective review of the project licence was considered by the AWERB on 19th September 2019 as part of the in-house assurance process. The retrospective review report was approved by the AWERB, and ongoing research supported by the committee.\n\nAll efforts were made to ameliorate suffering of animals throughout. To this end, the health and behaviour of all animals was checked at least twice daily, and animals were monitored remotely via webcams. Bodyweight was monitored daily. Humane endpoints included weight loss of >20% and adverse behavioural changes, such as signs of grimace (Langford et al., 2010), which did not improve following a 6h observation period.\n\nAdult, male C57BL/6J mice were obtained from an in-house colony maintained by the University of Oxford Biomedical Services (N=8, aged ~11 weeks at the start of the study). C57BL/6J was chosen as they are a common strain used for behavioural studies, or as a background for genetic mutants. Power calculations were not used for this study, as it was an exploratory study, and we expected that the effect size would be large due to a torpor bout typically involving a considerable (>5°C) drop in body temperature that is easily detected (Huang et al., 2021; van der Vinne et al., 2020). Furthermore, there are few existing studies of a similar nature which limits the ability to accurately perform power calculations.\n\nA single sex was used to maximise the applicability of the data to existing data, as most research to date, unfortunately, has been only conducted in males, especially within the behaviour field. Although we have not performed this study in females to date, we would expect similar outcomes due to torpor being readily induced in female mice elsewhere (Zhang et al., 2020). Mice were individually housed in wire-top cages (48×15×13 cm) on a 12:12 h light-dark cycle for the duration of the experiment. Cages were housed in custom light-tight chambers (LTCs; Figure 1A), with four cages per chamber (Fisher et al., 2012; Tam et al., 2021). LTCs were illuminated by cool white LED strips (Maplin, UK; 200 lux at the cage floor) during the light phase. Ambient room temperature and relative humidity were maintained at 21±1°C and 60±1%, respectively. Water was provided ad libitum throughout.\n\n(A) Mice were individually housed in standard M3 wire-top cages and placed under thermal imaging cameras (two cages per camera) which were used to continuously monitor environmental and skin temperature. Cameras were placed ~20 cm above the cage floor and positioned to ensure mice were in view. During food restriction, mice were fed at the same time each day and weighed ~2 hours after feeding. Mice were maintained at ~85% of free feeding bodyweight. (B) Thermal images of a mouse during euthermia (left), and a mouse during torpor (right). Skin temperature (Tskin) was measured by recording the warmest pixel in view every 1 s (1 Hz). Ta represents measurement of the ambient temperature (coldest pixel every 1 s). (C) Representative Tskin trace of a torpor bout during food restriction. Torpor bouts were detected by determining when Tskin had decreased by >3 standard deviation below the median euthermic temperature, for >1 h, for each mouse. Red circles indicate where torpor was determined to begin, and black circles indicate where torpor was determined to end using these criteria.\n\nThe change in peripheral skin temperature (Tskin) in response to food restriction was recorded using continuous thermal imaging cameras, mounted ~20 cm above the cage base (Optris® Xi 80 compact spot finder thermal imaging camera with 80° wide angle lens, Optris GmbH, Berlin, Germany; one camera per two animals). Custom Perspex blocks (23×12×3.2 cm, Aquarius Plastics, Surrey, UK) were used to block access to the area beneath the food hopper to minimise mice going out of view of the camera. Additionally, bedding material (sizzle nest) was kept to a minimum, and enrichment items, such as tunnels, were removed to prevent mice from being obscured from view of the thermal cameras, although ~10 g of nesting material was provided to align with the thermal preference of mice (Gaskill et al., 2011).\n\nTskin was determined by recording the hottest pixel every 1s for the duration of the study using the manufacturer’s software (Optris® PIX Connect, Optris GmbH). Data were processed by applying a high-pass filter 18°C and a low-pass filter at 35°C to remove artefacts that occurred due to movement and changes in animal posture. The filtered data were binned into 1-minute intervals, and a 20-minute moving average applied to smooth the data and further remove noise ready for analysis (Huang et al., 2021; van der Vinne et al., 2020).\n\nDuring this study, the effect of time of feeding on torpor characteristics were also assessed. As such, mice were assigned to one of two experimental groups: the morning-fed group and the night-fed group (n=4 for each). However, these data are beyond the scope of this report and will be described in an accompanying publication (Wilcox et al., 2024d). The analysis presented here are from all mice (n=8) combined. No criteria were set for including or excluding animals other than the humane endpoints, which was not reached by any animals.\n\nFollowing single housing, cages were placed in the LTCs, and mice left undisturbed for 2 weeks under ad libitum conditions to habituate to the new environment. Baseline measurements of bodyweight and Tskin were taken at the end of the habituation period prior to the start of food restriction. Food restriction was initiated by removing all food. A single ration of food (2.0-2.5g; 2016 Teklad Global Rodent Diet®, 16% protein, Envigo, Blackthorne, UK) was provided at the same time each day; the initial amount was determined by calculating 70% of individual average ad libitum daily intake, and then titrated based on bodyweight (van der Vinne et al., 2018). Mice were maintained at ~85% of their free feeding bodyweight, determined by taking a daily bodyweight measurement ~2 hours after food was provided. Additional food was provided the following day if bodyweight had dropped below 85%. Conversely, the following day’s food ration was reduced if bodyweight was >85%.\n\nTorpor induction was determined using non-invasive Tskin measurements. Previously we have shown that thermal imaging is able to reliably detect bouts of hypothermia associated with torpor (van der Vinne et al., 2020). However, Tskin cannot be used to calculate core body temperature (Tcore), making previously used torpor definitions of a Tcore <32°C inappropriate (Brown & Staples, 2010; Solymár et al., 2015; Swoap & Gutilla, 2009). Instead, we developed a custom algorithm, based on the criteria described by Huang et al. (2021), to define torpor as when Tskin dropped by >3 standard deviations below the median euthermic Tskin for >1h for each animal. The start and end of torpor were determined as when Tskin crossed this threshold (Figure 1C). These measurements were used to determine the effect of food restriction on torpor propensity and the amount of time spent in torpor.\n\nData were processed using MATLAB (MathWorks®, USA, v2023a) and analysed using Prism (GraphPad, version 9). Data are presented as mean values ± standard error of the mean (SEM) unless otherwise stated. Data were tested for normality using a Shapiro-Wilk test prior to analysis. One-way ANOVAs with repeated measures were used to determine how variables changed over time, followed by a post-hoc multiple comparisons test (Tukey’s) with a Bonferroni correction for multiple comparisons. A Geisser-Greenhouse correction was applied in all cases. Simple linear regression analysis was used to investigate the relationship between variables of interest. Data was determined to be significant when p<0.05.\n\n\nResults\n\nAs expected, food restriction resulted in a significant decrease in bodyweight over time (F(4.11, 28.8)=105.9, p<0.0001). Bodyweight dropped until day 8 of food restriction when weight stabilised between 85-90%, as was our aim in line with most behavioural food restriction paradigms (Figure 2A). To determine the effect of food restriction on body temperature, the change in mean Tskin from baseline (day 0, the last day of free feeding) was calculated. This method was chosen due to inter-individual variability in absolute Tskin measurements (van der Vinne et al., 2020). Tskin significantly decreased over days of food restriction, with the mean daily temperature dropping by ~3°C towards the end of the study (F(3.43,24.0)=34.9, p<0.0001; Figure 2B). The drop in Tskin is likely due to the induction of torpor, and mice spending more time in torpor. This is supported by an increasing proportion of mice entering torpor between days 1–7 of food restriction, and the increasing amount of time spent in torpor each day (Figure 2C, 2F). From day 8 onwards, all mice were entering at least one torpor bout per day. Further, the distribution of Tskin measurements prior to food restriction was unimodal, indicating that body temperature was being maintained with little deviation from a setpoint, as expected for endothermic species. However, after ~1 week of food restriction the distribution shifted to bimodal, with a second peak at a much lower Tskin, corresponding to heterothermy (Figure 2G).\n\n(A) Change in mean daily Tskin from baseline (day 0) over days of food restriction. Tskin was found to significantly decrease over days of food restriction (F(3.43,24.0)=34.9, p<0.0001; one-way ANOVA with repeated measures) with a maximal decrease of 3°C. Data are represented as a mean value ± SEM (n=8). (B) Bodyweight, expressed as a percentage of baseline (day 0), significantly decreased over days of food restriction, as expected (F(4.11,28.8)=105.9, p<0.0001; one-way ANOVA with repeated measures). Bodyweight stabilised at 85-90% on day 8 of food restriction. Data are represented as a mean value ± SEM (n=8). (C) Time course of the percentage of mice entering torpor on each day of food restriction. All mice reliably entered torpor each day from day 8 onwards (n=8) (D) Simple linear regression analysis of bodyweight (as a % of baseline) and change in body temperature compared to baseline (day 0). A strong positive relationship was found between the two parameters (r2=0.8989, p<0.0001). Each data point represents a day of recording. (E) A significant negative relationship was observed between the proportion of mice entering torpor and decrease of bodyweight (as a % of baseline). Note that all mice were entering torpor at 85% and 90% bodyweight, and almost all animals were entering torpor at slightly above 90% bodyweight. Each data point represents a day of recording. (F) The amount of time spent in torpor each day, expressed as a percentage, over days of food restriction. (G) Frequency distribution or recorded Tskin values from a representative mouse before food restriction (left), and during food restriction (right). A unimodal distribution of temperature is observed pre-food restriction, indicating maintenance around a set point. Two peaks are observed during food restriction, indicating a deviation from the euthermic set point, as is typical for heterothermy.\n\nNext, we investigated whether our observations were related to decreasing body weight. A strong positive relationship was found between bodyweight and the change in Tskin, with Tskin decreasing as bodyweight dropped (r2=0.8989, p<0.0001; Figure 2D). Moreover, a strong negative relationship was found between bodyweight and the proportion of mice entering torpor on each day of food restriction (r2=0.8725, p<0.0001). Notably, a high proportion of mice were entering torpor when bodyweight was at ~90% of free feeding bodyweight, indicating that even a weight loss of 10% is sufficient to induce torpor in laboratory mice (Figure 2E).\n\n\nDiscussion\n\nOur results show that torpor is readily induced in response to food restriction, typically occurring in all mice from day 8, although a high proportion of mice began entering torpor before this point. Notably, day 8 coincides with when bodyweight first began to stabilise at 85% bodyweight, which is often the target bodyweight required before behavioural testing commences (Pioli et al., 2014). Although bodyweight in this study was titrated to 85%, we still observed a high occurrence of torpor at bodyweights of 90%. Bodyweight can often increase to 90% during chronic food restriction paradigms; however, the data presented here suggest that even this degree of weight loss would be sufficient to induce torpor. As such, it is likely that torpor will be occurring regularly during the period of behavioural testing. Although it is unlikely that testing would be performed in torpid animals, as they are lethargic and show reduced behavioural responsiveness at low body temperatures, the short-term and delayed effects of torpor induction on brain function and behaviour are largely unknown. More generally, due to the profound changes in physiology associated with torpor, it is possible that variables of interest will be confounded due to previous torpor history. Although limited, some behavioural data gathered from post-torpid mice found that behavioural performance was significantly altered (Nowakowski et al., 2009). Moreover, a study in ground squirrels reported high levels of variability between individuals in memory retention when performing a previously learned task following torpor (Hensleigh et al., 2022).\n\nHere, we focused on a chronic food restriction paradigm at ~85% of free-feeding bodyweight; however, previous work has shown that torpor can be induced after only 7 hours of total food removal (Brown & Staples, 2010; Swoap et al., 2006; Swoap & Weinshenker, 2008). Acute fasting is commonplace as part of metabolic and pharmacological studies (Ayala et al., 2010; Beumer et al., 2006), therefore, based on previous research, we anticipate that our findings are also applicable to studies where acute fasting is used.\n\nDue to technical limitations of the thermal cameras, mice were singly housed throughout this study. Further, single housing allowed for more precise titration of the amount of food provided each day to maintain individual bodyweight at ~85%. Single housing is common practice during torpor studies in mice due to the use of thermal cameras (Hitrec et al., 2019), or due to surgery being required to implant temperature-sensitive telemeters (Oelkrug et al., 2011). However, group housing is often used during behavioural testing to reduce cage costs and for welfare benefits. There is some evidence in other species that torpor is still employed in group settings and can result in increased entries into torpor due to reduced energy savings because of social thermoregulation via huddling (Geiser, 2010; Jefimow et al., 2011). Moreover, a study in group housed sugar gliders found that food restriction resulted in synchronisation of torpor bouts (Nowack & Geiser, 2016). To our knowledge, social torpor has not been investigated in laboratory mice; however, evidence from other species suggests that the results presented here would be applicable to group housed mice.\n\nBased on our data, we recommend that studies using both acute and long-term food restriction in mice are closely monitored for torpor bouts, for example, using continuous non-invasive thermal imaging. Even in group housed settings, thermal imaging could be used to detect sustained drops in body temperature corresponding to mice entering torpor. Doing so will enable data to be compared from cages with similar torpor histories before running a task or taking a sample. Moreover, knowing prior torpor history will allow for additional controls to be implemented and may help to explain unexpected differences between experimental units. From a 3Rs perspective, our recommendations can be used to refine experimental paradigms requiring food restriction, therefore helping to reduce variability.\n\n\nAuthor contributions\n\nSLW, SNP, DMB and VVV designed experiments. SLW performed measurements. SLW and VVV performed analysis. SLW wrote the manuscript with input from all other authors.", "appendix": "Data availability\n\nFigshare: Induction of torpor in response to a common chronic food restriction paradigm: implications for behavioural research using mice.\n\n- Bodyweight measurements: https://doi.org/10.6084/m9.figshare.25722546 (Wilcox et al., 2024a).\n\n- Temperature recordings: https://doi.org/10.6084/m9.figshare.25722426 (Wilcox et al., 2024b).\n\nThis project contains the following underlying data:\n\n- Bodyweight.xlsx\n\n- 230920_1.dat\n\n- 230920_2.dat\n\n- 230920_3.dat\n\n- 230920_4.dat\n\n- 240920_1.dat\n\n- 240920_2.dat\n\n- 240920_3.dat\n\n- 240920_4.dat\n\n- 250920_1.dat\n\n- 250920_2.dat\n\n- 250920_3.dat\n\n- 250920_4.dat\n\n- 260920_1.dat\n\n- 260920_2.dat\n\n- 260920_3.dat\n\n- 260920_4.dat\n\n- 270920_1.dat\n\n- 270920_2.dat\n\n- 270920_3.dat\n\n- 270920_4.dat\n\n- 280920_1.dat\n\n- 280920_2.dat\n\n- 280920_3.dat\n\n- 280920_4.dat\n\n- 290920_1.dat\n\n- 290920_2.dat\n\n- 290920_3.dat\n\n- 290920_4.dat\n\n- 300920_1.dat\n\n- 300920_2.dat\n\n- 300920_3.dat\n\n- 300920_4.dat\n\n- 011020_1.dat\n\n- 011020_2.dat\n\n- 011020_3.dat\n\n- 011020_4.dat\n\n- 021020_1.dat\n\n- 021020_2.dat\n\n- 021020_3.dat\n\n- 021020_4.dat\n\n- 031020_1.dat\n\n- 031020_2.dat\n\n- 031020_3.dat\n\n- 031020_4.dat\n\n- 041020_1.dat\n\n- 041020_2.dat\n\n- 041020_3.dat\n\n- 041020_4.dat\n\n- 051020_1.dat\n\n- 051020_2.dat\n\n- 051020_3.dat\n\n- 051020_4.dat\n\n- 061020_1.dat\n\n- 061020_2.dat\n\n- 061020_3.dat\n\n- 061020_4.dat\n\n- 071020_1.dat\n\n- 071020_2.dat\n\n- 071020_3.dat\n\n- 071020_4.dat\n\n- 081020_1.dat\n\n- 081020_2.dat\n\n- 081020_3.dat\n\n- 081020_4.dat\n\n- 091020_1.dat\n\n- 091020_2.dat\n\n- 091020_3.dat\n\n- 091020_4.dat\n\n- 101020_1.dat\n\n- 101020_2.dat\n\n- 101020_3.dat\n\n- 101020_4.dat\n\n- 121020_1.dat\n\n- 121020_2.dat\n\n- 121020_3.dat\n\n- 121020_4.dat\n\n- 131020_1.dat\n\n- 131020_2.dat\n\n- 131020_3.dat\n\n- 131020_4.dat\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: ARRIVE author checklist: https://doi.org/10.6084/m9.figshare.26213432 (Wilcox, 2024c)\n\n- ARRIVE Author Checklist - BW and torpor.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Laura E. McKillop and Vincent van der Vinne for assistance during the planning and running of experiments. We thank Sara Wells for her comments on this manuscript.\n\n\nReferences\n\nAmbler M, Hitrec T, Pickering A: Turn it off and on again: Characteristics and control of torpor. Wellcome Open Research. 2021; 6: 313. Publisher Full Text\n\nAnnual Statistics of Scientific Procedures on Living Animals Great Britain: 2021.\n\nAyala JE, Samuel VT, Morton GJ, et al.: Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice. Dis. Model. Mech. 2010; 3(9-10): 525–534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker M, Penny D: Is there a reproducibility crisis? Nature. 2016; 533(7604): 452–454. PubMed Abstract \| Publisher Full Text\n\nBank JHH, Kemmling J, Rijntjes E, et al.: Thyroid hormone status affects expression of daily torpor and gene transcription in Djungarian hamsters (Phodopus sungorus). Horm. Behav. 2015; 75: 120–129. PubMed Abstract \| Publisher Full Text\n\nBegley CG, Ioannidis JPA: Reproducibility in Science. Circ. Res. 2015; 116(1): 116–126. Publisher Full Text\n\nBerglund ED, Li CY, Poffenberger G, et al.: Glucose metabolism in vivo in four commonly used inbred mouse strains. Diabetes. 2008; 57(7): 1790–1799. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeumer JH, Eiseman JL, Parise RA, et al.: Pharmacokinetics, Metabolism, and Oral Bioavailability of the DNA Methyltransferase Inhibitor 5-Fluoro-2 -Deoxycytidine in Mice. Clin. Cancer Res. 2006; 12(24): 7483–7491. PubMed Abstract \| Publisher Full Text\n\nBrown JCL, Staples JF: Mitochondrial metabolism during fasting-induced daily torpor in mice. Bioenergetics. 2010; 1797: 476–486. PubMed Abstract \| Publisher Full Text\n\nChen L, Zhang B, Liu J, et al.: Pharmacokinetics and bioavailability study of monocrotaline in mouse blood by ultra-performance liquid chromatography-tandem mass spectrometry. Biomed. Res. Int. 2018; 2018: 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFisher SP, Godinho SIH, Pothecary CA, et al.: Rapid assessment of sleep-wake behavior in mice. J. Biol. Rhythm. 2012; 27(1): 48–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaskill BN, Rohr SA, Pajor EA, et al.: Working with what you’ve got: Changes in thermal preference and behavior in mice with or without nesting material. J. Therm. Biol. 2011; 36(3): 193–199. Publisher Full Text\n\nGavrilova O, Leon LR, Marcus-Samuels B, et al.: Torpor in mice is induced by both leptin-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. USA. 1999; 96(25): 14623–14628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeiser F: Hibernation, Daily Torpor and Estivation in Mammals and Birds: Behavioral Aspects.Breed MD, Moore J, editors. Encyclopedia of Animal Behavior. Oxford: Academic Press; 2010; Vol. 2. : pp. 77–83. Publisher Full Text\n\nGluck EF, Stephens N, Swoap SJ: Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006; 291(5): R1303–R1309. PubMed Abstract \| Publisher Full Text\n\nGreenwell BJ, Trott AJ, Beytebiere JR, et al.: Rhythmic Food Intake Drives Rhythmic Gene Expression More Potently than the Hepatic Circadian Clock in Mice. Cell Rep. 2019; 27(3): 649–657.e5. PubMed Abstract \| Publisher Full Text\n\nHensleigh E, Murtishaw AS, Treat MD, et al.: Torpor Does Not Influence Spatial Memory in Hibernating Golden-Mantled Ground Squirrels (Spermophilus [Callospermophilus] lateralis). Physiol. Biochem. Zool. 2022; 95(5): 390–399. PubMed Abstract \| Publisher Full Text\n\nHitrec T, Luppi M, Bastianini S, et al.: Neural control of fasting-induced torpor in mice. Sci. Rep. 2019; 9(1): 15462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang H, Yang Y, Lv C, et al.: Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin pill following oral administration to mice. J. Ethnopharmacol. 2015; 161: 175–185. PubMed Abstract \| Publisher Full Text\n\nHuang YG, Flaherty SJ, Pothecary CA, et al.: The relationship between fasting-induced torpor, sleep, and wakefulness in laboratory mice. Sleep. 2021; 44(9): 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHudson JW, Scott IM: Daily torpor in the laboratory mouse, mus musculus var. albino. Physiol. Zool. 1979; 52(2): 205–218. Publisher Full Text\n\nJefimow M, Gła̧bska M, Wojciechowski MS: Social thermoregulation and torpor in the Siberian hamster. J. Exp. Biol. 2011; 214(7): 1100–1108. PubMed Abstract \| Publisher Full Text\n\nJensen TL, Kiersgaard MK, Sørensen DB, et al.: Fasting of mice: A review. Lab. Anim. 2013; 47(4): 225–240. Publisher Full Text\n\nKato GA, Sakamoto SH, Eto T, et al.: Individual differences in torpor expression in adult mice are related to relative birth mass. J. Exp. Biol. 2018; 221. PubMed Abstract \| Publisher Full Text\n\nLangford DJ, Bailey AL, Chanda ML, et al.: Coding of facial expressions of pain in the laboratory mouse. Nat. Methods. 2010; 7(6): 447–449. PubMed Abstract \| Publisher Full Text\n\nMandillo S, Tucci V, Hölter SM, et al.: Reliability, robustness, and reproducibility in mouse behavioral phenotyping: A cross-laboratory study. Physiol. Genomics. 2008; 34(3): 243–255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNortheast RC, Huang Y, McKillop LE, et al.: Sleep homeostasis during daytime food entrainment in mice. Sleep. 2019; 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNowack J, Geiser F: Friends with benefits: The role of huddling in mixed groups of torpid and normothermic animals. J. Exp. Biol. 2016; 219(4): 590–596. PubMed Abstract \| Publisher Full Text\n\nNowakowski SG, Swoap SJ, Sandstrom NJ: A single bout of torpor in mice protects memory processes. Physiol. Behav. 2009; 97(1): 115–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOelkrug R, Heldmaier G, Meyer CW: Torpor patterns, arousal rates, and temporal organization of torpor entry in wildtype and UCP1-ablated mice. J. Comp. Physiol. B. 2011; 181(1): 137–145. PubMed Abstract \| Publisher Full Text\n\nPadamsey Z, Katsanevaki D, Dupuy N, et al.: Neocortex saves energy by reducing coding precision during food scarcity. Neuron. 2021; 110: 280–296.e10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPioli EY, Gaskill BN, Gilmour G, et al.: An automated maze task for assessing hippocampus-sensitive memory in mice. Behav. Brain Res. 2014; 261: 249–257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolymár M, Pétervári E, Balaskó M, et al.: The onset of daily torpor is regulated by the same low body mass in lean mice and in mice with diet-induced obesity. Temperature. 2015; 2(1): 129–134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwoap SJ, Gutilla MJ: Cardiovascular changes during daily torpor in the laboratory mouse. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 297(3): R769–R774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwoap SJ, Gutilla MJ, Liles LC, et al.: The Full Expression of Fasting-Induced Torpor Requires 3-Adrenergic Receptor Signaling. J. Neurosci. 2006; 26(1): 241–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwoap SJ, Weinshenker D: Norepinephrine Controls Both Torpor Initiation and Emergence via Distinct Mechanisms in the Mouse. PLoS One. 2008; 3(12): e4038. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTam SKE, Brown LA, Wilson TS, et al.: Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and short-term memory. PNAS. 2021; 118(39): e2101591118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Vinne V , Bingaman MJ, Weaver DR, et al.: Clocks and meals keep mice from being cool. J. Exp. Biol. 2018; 221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Vinne V , Pothecary CA, Wilcox SL, et al.: Continuous and non-invasive thermography of mouse skin accurately describes core body temperature patterns, but not absolute core temperature. Sci. Rep. 2020; 10(1): 20680–20689. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVyazovskiy V v, Palchykova S, Achermann P, et al.: Different Effects of Sleep Deprivation and Torpor on EEG Slow-Wave Characteristics in Djungarian Hamsters. Cereb. Cortex. 2017; 27(2): 950–961. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilcox S, et al.: Bodyweight measurements. Dataset. figshare. 2024a. Publisher Full Text\n\nWilcox S, et al.: Body temperature recordings. Dataset. figshare. 2024b. Publisher Full Text\n\nWilcox S: ARRIVE guidelines author checklist. Dataset. figshare. 2024c. Publisher Full Text\n\nWilcox S, et al.: The effect of food timing on torpor propensity and characteristics in laboratory mice during a common food restriction paradigm. [version 1; peer review: awaiting peer review]. F1000Res. 2024d. Publisher Full Text\n\nZhang Z, Reis FMCV, He Y, et al.: Estrogen-sensitive medial preoptic area neurons coordinate torpor in mice. Nat. Commun. 2020; 11(1): 6314–6378. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "329625", "date": "22 Oct 2024", "name": "Timna Hitrec", "expertise": [ "Reviewer Expertise Hibernation", "torpor", "physiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWilcox and colleagues show that chronic food restriction to 85% of ad libitum intake leads to repeated bouts of torpor in male mice. A significant strength of this study lies in highlighting a crucial, often overlooked consideration: the occurrence of torpor episodes, which should be accounted for and controlled in experimental designs involving food restriction in mice. I have a few minor observations:\nThe authors mention dividing the animals into two groups fed at different times of the day. I wonder if it’s appropriate to merge these groups without evidence showing they are not significantly different in terms of torpor duration and depth? Also, could the authors provide the specific ZT feeding times? This detail would be beneficial for researchers planning food restriction experiments. Based on my experience, initial weight can significantly influence torpor occurrence. Could the authors provide the absolute mean starting weights of the animals, as well as their final weights at the end of the experiment? In Figure 1C, could the axes be graduated to display absolute temperature values? This would enhance readability, and a larger font size would also help. It appears that two figures have been swapped. At the beginning of the results section, the authors discuss body weight reduction and reference Figure 2A, which shows Tskin. Conversely, when discussing Tskin, they reference Figure 2B, which displays body weight changes. Please clarify whether the means shown in Figure 2 refer to the entire 24-hour period for the indicated 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? 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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "329627", "date": "25 Oct 2024", "name": "Cara Green", "expertise": [ "Reviewer Expertise Nutritional interventions", "ageing", "metabolism" ], "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\nInduction of torpor in response to a common chronic food restriction paradigm: implications for behavioural research using mice\nWilcox and colleagues detail how food restriction to decrease body weight to 85% of baseline can effect torpor occurrence in male C57 mice. Their results are clear and it is useful for researchers to consider how torpor might impact behavioural experiments in mice. However, much more clarity of thought and discussion is required before this work is approved for indexing, as it could be clearer how this furthers what we currently know about food restriction and torpor.\nThis study only used males. Both sexes should be used unless a robust justification is given (as per the UKRI). “A single sex was used to maximise the applicability of the data to existing data” is not a robust justification. This study does not need to be applicable to research that has already been completed and published. This research, which is generated to help with study designs and future work should contain both male and female mice. This should be repeated in female mice to have a broader applicability. “Although we have not performed this study in females to date, we would expect similar outcomes due to torpor being readily induced in female mice elsewhere”. Studies have shown that female mice are more prone to the induction of torpor. This should be mentioned.\n\nAs you rightly point out bedding and enrichment is kept to a minimum for the benefit of your thermal imaging. Is this relevant to long term studies where more bedding and enrichment is necessary. Would a different method of body temperature measurement be more relevant? Please add to discussion. Why did you use mice of this age? Mice were kept at 85% of their free feeding body weight at 12/13 weeks of age. Mice gain weight over time and are not necessarily fully developed at 12 weeks. Can you comment on whether reducing food intake at this age may be hindering development and therefore confounding your results? Please add to discussion. Is there a scientific reason for using 3 SDs to define torpor in your methods? If you could provide clarity that would be helpful. “Acute fasting is commonplace as part of metabolic and pharmacological studies (Ayala et al., 2010; Beumer et al., 2006), therefore, based on previous research, we anticipate that our findings are also applicable to studies where acute fasting is used.” It may be true that acute fasting can also promote torpor and affect others work, but you have shown no hourly data therefore I don’t think you can claim that your work is applicable. In figure 1B your camera images show a 9°C difference between euthermia and torpor but we do not get to see results like this in the rest of the MS. I think it would be interesting/helpful to graph the change in highest to lowest body temperature in a 24 hour period for each mouse over time in figure 2 (as well as using baseline Tskin as you have done). Could you (possibly in supplementary) also graph the raw Tskin values for the graphs in Figure 2. Especially for researchers looking to replicate this work, I think it would be useful to know what kinds of skin temperatures you are seeing with your thermal camera set up. It is possible I missed this but how long were you measuring mice with thermal camera for? What time of day. Was each day measured for the whole 24 hour period? Can you possibly plot changes over the day (similar to point 7)? This would show if some mice were some mice going into torpor/arousal more quickly and the relationship with body weight. “A single ration of food provided at the same time each day”. In the previous statement you just told us you had night and morning fed mice so this must be false. How long were mice in the study once diets were switched I can’t see this stated, sorry if I missed it. Work similar to this has been done by the Speakman Lab, but is not mentioned in the introduction. I think this is quite a large oversight. They looked at different levels of CR started at 5 months of age on torpor incidence. Due to the age of the mice, I suspect torpor was not seen as readily. This may be worth adding to your discussion.\nMitchell SE et al. (2015 [Ref - 1]) https://pmc.ncbi.nlm.nih.gov/articles/PMC4621893/ Mitchell SE et al. (2015 [Ref - 2]) https://pmc.ncbi.nlm.nih.gov/articles/PMC4599246/\n\n13. It’s great that you’ve provided source material, however, to be actually useful I think you need to collate it into one excel file with clearer headings etc. As it stands it’s pretty un-user friendly.\n14. This is just a PSA, could you add line numbers in the future to your papers when you submit? It helps the reviewers a lot!\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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-830
https://f1000research.com/articles/13-829/v1	24 Jul 24	{ "type": "Brief Report", "title": "The effect of food timing on torpor propensity and characteristics in laboratory mice during a common food restriction paradigm", "authors": [ "Sian L. Wilcox", "David M. Bannerman", "Stuart N. Peirson", "Vladyslav V. Vyazovskiy", "David M. Bannerman", "Stuart N. Peirson", "Vladyslav V. Vyazovskiy" ], "abstract": "Background Many behavioural, pharmacological, and metabolic studies in mice require fasting, yet the possibility of fasting-induced torpor affecting the data is rarely considered. Torpor is a state characterised by depressed metabolism and profound alterations to physiology and behaviour. In this study we aimed to determine how the effects of torpor on experimental outcomes could be mitigated.\n\nMethods To this end, timing and characteristics of fasting-induced torpor in response to feeding in the morning versus feeding in the night were compared using non-invasive monitoring of peripheral body temperature.\n\nResults Night-fed mice entered significantly more torpor bouts per day compared to morning-fed mice (Morning: 2.79±0.197 (mean ± SEM); Night: 4.79±0.533 (mean ± SEM); p=0.0125), but these bouts were shorter on average by ~1.5h. Latency to the first torpor bout following feeding tended to be shorter during night feeding (Morning: 9.57±0.8h (mean ± SEM); Night: 6.66±1.2h (mean ± SEM); p=0.0928). Moreover, torpor bouts typically occurred during the dark phase in the morning-fed group, whilst night feeding resulted in a shift of torpor occurrence to earlier in the day (Morning: 14.2±0.4 ZT h (mean ± SEM); Night: 12.2±0.9 ZT h (mean ± SEM); p=0.0933). There was a high degree of variation in torpor occurrence within and between animals in each group.\n\nConclusions We recommend that feeding time is kept consistent between days and the same across animals to minimise variation in torpor occurrence. Moreover, the timing of food provision may be optimised to allow measurements to be taken during euthermia, to mitigate the effects of torpor on the variables investigated. Finally, we recommend that body temperature is monitored non-invasively to determine when torpor is occurring, and that testing, or sample collection is conducted when the torpor history is comparable between animals.", "keywords": [ "3Rs", "Daily torpor", "Food restriction", "Mice", "Circadian", "Refinement" ], "content": "\n\n\n\nScientific benefit:\n\n\n\n• Identification of the effects of feeding schedule on torpor propensity and characteristics in laboratory mice\n\n3Rs benefit:\n\n\n\n• Identifying and controlling for potential sources of variation in experimental variables due to torpor occurrence\n\n• More accurate and precise induction of torpor in mice thereby reducing the time spent on food restriction and shortening the duration of stress\n\n• Use of food restriction to study torpor and the medium-long term physiological impacts of torpor, mean that the use of cross-over studies need to be carefully designed/considered to factor in these additional variables\n\nCurrent applications:\n\n\n\n• Research using food restriction techniques in mice, particularly neurobiology of hypometabolism and circadian phenotyping\n\nPotential applications:\n\n\n\n• Research fields which regularly use food restriction in mice e.g., metabolic studies, behavioural research, pharmacological studies\n\n\nIntroduction\n\nFood restriction and fasting are common techniques used in behavioural, metabolic, and circadian studies, amongst others. However, many of these studies do not account for the induction of torpor in response to limited food availability in mice. Torpor is a hypometabolic and hypothermic state employed by certain species to conserve energy, but it also profoundly alters central (Huang et al., 2021; von der Ohe et al., 2006) and peripheral (Melvin & Andrews, 2009) physiology. Laboratory mice readily enter torpor in response to fasting (Hudson & Scott, 1979; Swoap et al., 2006) but the long-term effects of chronic torpor induction on subsequent behaviour and physiology are largely unknown.\n\nFood timing is rarely reported in studies using food restriction, and feeding schedule may vary depending on when a task is performed, or sample taken. However, torpor propensity has been shown to be under circadian control and is strongly influenced by food timing (Oelkrug et al., 2011; van der Vinne et al., 2018). In an accompanying publication (Wilcox et al., 2024d), we reported that torpor was readily induced during a chronic food restriction paradigm and that the likelihood of torpor occurrence was closely related to decreased bodyweight. Subsequently, we hypothesised that differences in food timing within and between studies may result in variation in torpor characteristics and contribute to variations in physiology and behaviour due to differences in prior torpor history. Here, we investigated the effect of food timing on torpor characteristics. All mice studied (n=8) readily entered torpor in response to food restriction, regardless of feeding time, and feeding time significantly affected the latency to torpor, timing, and duration of torpor bouts.\n\n\nMethods\n\nProcedures were performed in compliance with the United Kingdom Animals (Scientific Procedures) Act of 1986 (Project Licence Number: P828B64BC). All experiments were approved by the University of Oxford Animal Welfare and Ethical Review Board (AWERB) on 28th February 2017 (reference: DPAGEP (17)9, Item 5a). A retrospective review of the project licence was considered by the AWERB on 19th September 2019 as part of the in-house assurance process. The retrospective review report was approved by the AWERB, and ongoing research supported by the committee.\n\nAll efforts were made to ameliorate suffering of animals throughout. To this end, the health and behaviour of all animals was checked at least twice daily, and animals were monitored remotely via webcams. Bodyweight was monitored daily. Humane endpoints included weight loss of >20% and adverse behavioural changes, such as signs of grimace (Langford et al., 2010), which did not improve following a 6h observation period.\n\nC57BL/6J mice were used in this study (University of Oxford Biomedical Services in-house colony; N=8, aged ~11 weeks). Male mice were chosen for this study to ensure data were representative of the current research landscape, which primarily uses males. However, we expect that these data will be applicable to females, as torpor has been shown to be readily induced in female mice (Oelkrug et al., 2011; Zhang et al., 2020). The C57BL/6J strain was chosen as they are commonly used for behavioural studies, or as a background for genetic mutants.\n\nMice were individually housed in wire-top cages (48x15x13cm) on a 12:12h light-dark (LD) cycle for the duration of the experiment. Cages were housed in custom light-tight chambers (LTCs; Figure 1B), with four cages per chamber (Fisher et al., 2012; Tam et al., 2021). LTCs were illuminated by cool white LED strips (Maplin, UK; 200 lux at the cage floor). Ambient room temperature and relative humidity were maintained at 21±1°C and 60±1%, respectively. Water was provided ad libitum throughout. Bedding material (sizzle nest) was kept to a minimum, and enrichment items, such as tunnels, were removed to prevent mice from being obscured from view of the thermal cameras, although ~10g of nesting material was provided to align with the thermal preference of mice (Gaskill et al., 2011).\n\n(A) Mice were assigned to either the morning-fed group, or the night-fed group (n=4). Night-fed animals were kept on a reverse light-dark cycle (lights on at 7 pm), to allow for feeding to be completed in parallel. During food restriction, all mice were fed once daily; morning-fed mice were fed one hour after lights on, whilst night-fed mice were fed one hour after lights off. Mice were weighed ~2 hours after food was provided; additional food was provided as necessary at this time to ensure bodyweight was maintained at ~85% of free feeding bodyweight. (B) Mice were individually housed in standard wire-top cages, placed under continuous thermal imaging cameras (two cages per camera), in light-tight chambers. Cameras were placed ~20 cm above the cage floor and positioned to ensure mice were in view. (C) Thermal images of a mouse during euthermia (left), and a mouse during torpor (right). Skin temperature was measured by recording the warmest pixel in view every 1 s (1 Hz). Ta represents measurement of the ambient temperature (coldest pixel every 1 s). (D) Representative skin temperature (Tskin) trace of a torpor bout during food restriction. Torpor bouts were detected by determining when Tskin had decreased by >3 standard deviation below the median euthermic temperature, for >1 h. Red circles indicate where torpor was determined to begin, and black circles indicate where torpor was determined to end using these criteria. (E) Raw Tskin temperature traces over consecutive days of food restriction (days 8–17) from a representative morning-fed mouse (left) and a representative night-fed mouse (right). Feeding time is indicated by the pink line, whilst the time at which mice were weighed is shown by the black line.\n\nMice were assigned to one of two groups: the morning-fed group and the night-fed group (n=4 for each), in a matched manner using starting bodyweight. Experimenters were not blinded to group during allocation or data collection.\n\nFollowing single-housing, mice were given 3 days to habituate to the LTCs in which the cages were housed. The morning-fed group and the night-fed group were housed in separate LTCs. The LD cycle for the night-fed group was shifted by one hour each day until a reverse LD cycle was achieved (lights on: 7 pm), taking a total of 12 days. During food restriction, the morning group received food one hour after lights on, at Zeitgeber Time 1 (ZT1), whereas the night group received food one hour after lights off, at ZT13. Due to the reverse LD cycle for the night-fed group, both groups were fed at the same real time (8 am). LTCs were opened for both feeding and weighing which was performed under dim red light for the night-fed group; LTCs were not opened in parallel to prevent light leakage from the morning-fed group’s LTC.\n\nDuring food restriction, a single ration of food (2.0-2.5 g, 2016 Teklad Global Rodent Diet®, 16% protein, Envigo, Blackthorne, UK) was provided at the same time each day; the initial amount was determined by calculating 70% of individual average ad libitum daily intake, and then titrated based on bodyweight (van der Vinne et al., 2018). Mice were maintained at ~85% of their free feeding bodyweight, determined by taking daily bodyweight measurements ~2 hours after feeding. Additional food was provided the following day if bodyweight had dropped below 85%. Conversely, the following day’s food ration was reduced if bodyweight was >85%.\n\nPower calculations were not used due to the exploratory nature of this study, and we expected that the effect size would be large due to a torpor bout typically involving a considerable (>5°C) drop in body temperature that is easily detected (Huang et al., 2021; van der Vinne et al., 2020). Furthermore, there are few existing studies of a similar nature which limits the ability to accurately perform power calculations. We had initially planned to reverse the group treatments to allow for a within-subjects design; however, food restriction had a profound effect on bodyweight which was found to be closely related to torpor induction. As such, we determined that the animal’s physiology had been altered to such a degree that comparing variables pre- and post-switch would be inappropriate. Whilst this highlights one of our key messages that previous experience of the animals, including torpor history, matters greatly, excluding half of the animals from further analysis does reduce the power of this study. Therefore, we consider these findings preliminary.\n\nPeripheral skin temperature (Tskin) was non-invasively recorded using continuous thermal imaging cameras, mounted ~20 cm above the cage base (Optris® Xi 80 compact spot finder thermal imaging camera with 80° wide angle lens, Optris GmbH, Berlin, Germany; one camera per two animals; Figure 1B). Custom Perspex blocks (23×12×3.2 cm, Aquarius Plastics, Surrey, UK) were used to block access to the area beneath the food hopper to minimise mice going out of view of the camera during recording.\n\nTskin was determined by recording the hottest pixel every 1s for the duration of the study using the manufacturer’s software (Optris® PIX Connect, Optris GmbH). Data were processed by applying a high-pass filter 18°C and a low-pass filter at 35°C to remove artefacts that occurred due to movement and changes in animal posture. The filtered data were binned into 1-minute intervals, and a 20-minute moving average applied to smooth the data and further remove noise ready for analysis (Huang et al., 2021; van der Vinne et al., 2020). Tskin measurements were used to determine torpor characteristics, including latency to torpor, the number of bouts, and torpor timing.\n\nThermal imaging can reliably detect bouts of hypothermia associated with torpor (van der Vinne et al., 2020), but cannot be used to calculate core body temperature (Tcore), making previously used torpor definitions of a Tcore <32°C inappropriate (Brown & Staples, 2010; Solymár et al., 2015; Swoap & Gutilla, 2009). Instead, we developed a custom algorithm, based on the criteria described by Huang et al. (2021), to define torpor as when Tskin dropped by >3 standard deviations below the median euthermic Tskin for >1h for each animal. The start and end of torpor were determined as when Tskin crossed this threshold (Figure 1D).\n\nThe data were first blinded prior to processing and analysis. Data were processed using MATLAB (MathWorks®, USA, v2023a) and analysed using Prism (GraphPad, version 9). Data are presented as a group mean ± standard error of the mean, with data from all four mice per group for each analysis. No criteria were set for including or excluding animals, other than the humane endpoints; no animals were excluded from analysis. Data were tested for normality using a Shapiro-Wilk test prior to analysis. Two-way ANOVAs with repeated measures were used to determine the effect of time and group on torpor characteristics. A Geisser-Greenhouse correction was used in all cases. Between group comparisons were performed on the final 6 days of food restriction using unpaired t-tests, as this was the point at which parameters of interest had stabilised.\n\n\nResults\n\nThe change in daily mean Tskin compared to baseline (day 0, the last day of ad libitum feeding) was calculated and used for comparisons due to absolute Tskin values varying between individuals (van der Vinne et al., 2020). Tskin decreased over days of food restriction as mice spent more time in torpor (Figure 2A-B). Day of food restriction significantly affected Tskin in both groups (F(3.04,18.3)=32.3, p<0.0001), but no significant group effect was found (F(1,6)=0.413, p=0.5441), nor was there a significant interaction between time and group (F(17,102)=0.4898, p=0.9525).\n\n(A) Change in mean daily Tskin, relative to baseline, over days of food restriction (mean ± SEM). Tskin significantly decreased as food restriction progressed for both groups (F(3.04,18.3)=32.3, p<0.0001; Two-way ANOVA with repeated measures). (B) Comparison of mean change in Tskin on the last 6 days of recording, when temperature and bodyweight decline had stabilised (mean ± SEM). There was no difference in change in Tskin between groups (p=0.5860, t(6)=0.5753; unpaired t-test). (C) Mean number of torpor bouts per day over days of food restriction. The number of daily bouts significantly increased during food restriction (F(3.15,18.9)=8.85, p=0.0006; Two-way ANOVA with repeated measures). (D) Comparison of the mean number of bouts entered by each group across the final 6 days of food restriction when bodyweight had stabilised (mean ± SEM). The night group entered significantly more torpor bouts per day compared to the morning group (p=0.0125, t(6)=3.52; unpaired t-test). (E) Change in the latency to the first torpor bout after feeding over days of food restriction (mean ± SEM). Latency to torpor decreased over time. (F) Comparison of latency to torpor in stabilised conditions (mean ± SEM). No difference was found between groups (p=0.0928, t(6)=1.997; unpaired t-test). (G) Comparison of relative torpor timing over the 24h recording period for the final 6 days of food restriction (mean ± SEM). On average, torpor bouts occurred later in the day during morning-feeding compared to the night group but this difference was not significant (p=0.0933, t(6)=1.99; unpaired t-test). (H) Change in the mean duration of torpor bouts over days of food restriction (mean ± SEM). Torpor bouts increased in length over time for both groups (F(3.06,18.4)=7.87, p=0.0013; Two-way ANOVA with repeated measures). (I) Comparison of mean torpor bout length on the final 6 days of recording (mean ± SEM). Morning-fed bouts tended to be longer on average (p=0.0558, t(6)=2.37; unpaired t-test). (J) Comparison of the longest bout per day over the final 6 days of food restriction (mean ± SEM). No difference was found between groups (p=0.4473, t(6)=0.813; unpaired t-test).\n\nRaw temperature traces suggested potential differences in torpor timing and length between groups (Figure 1E). Unsurprisingly, the number of torpor bouts increased as food restriction progressed before stabilising in line with bodyweight (F(3.15,18.9)=8.85, p=0.0006; Figure 2C). Feeding time was a significant source of variation, but there was no interaction between group and day of food restriction. Comparison when bodyweight stabilised found that the night group entered significantly more torpor bouts per day compared to the morning fed group (Morning: 2.8±0.2, 95% CI [2.17–3.42]; Night: 4.8±0.5, 95% CI [3.10–6.49]; t(6)=3.52, p=0.0125; Figure 2D).\n\nLatency to torpor was highly variable between animals up to day 8 of food restriction, although mean latency decreased, before stabilising from day 9 for both groups (Figure 2E). No significant group difference was found for stabilised latencies, although average latency was longer for the morning-fed group (Morning: 9.6±0.8h, 95% CI [6.91–12.2]; Night: 6.7±1.2h, 95% CI [2.87–10.5]; p=0.0928, t(6)=1.997; Figure 2F). Additionally, no statistical difference in the relative timing of torpor over each 24h recording period, although night-fed mice entered torpor earlier on average (Morning: 14.2±0.4 ZTh, 95% CI [13.1–15.3]; Night: 12.2±0.9 ZTh, 95% CI [9.23–15.2]; p=0.0933, t(6)=1.99; Figure 2G). However, torpor timing varied greatly in each group, especially in the night-fed group. As such, a significant difference may be found with a larger sample size.\n\nTorpor bouts increased in duration as food restriction progressed for both groups (F(3.06,18.4)=7.87, p=0.0013), before stabilising in line with bodyweight stabilisation at 85% (Figure 2H). On average, torpor bouts were longer in the morning-fed group compared to the night-fed group, and was trending towards statistical significance (Morning: 4.4±0.7h, 95% CI [2.02 6.72]; Night: 2.6±0.1h, 95% CI [2.16–3.03]; p=0.0558, t(6)=2.37; Figure 2I). There was no statistical difference in the length of the longest torpor bout per day between groups (Morning: 7.3±1.3h, 95% CI [3.08–11.5]; Night: 6.0±0.8h, 95% CI [3.51–8.55]; p=0.4473, t(6)=0.813; Figure 2J).\n\n\nDiscussion\n\nPreviously, we reported that torpor is readily induced in response to food restriction with reliable torpor bouts occurring daily when bodyweight reaches 85-90% of free feeding bodyweight (Wilcox et al., 2024d). Here, we investigated the effect of feeding time on torpor characteristics. We report that feeding time influenced some torpor characteristics, namely the number of torpor bouts being entered each day, with night feeding resulting in more torpor bouts. Although bout duration was not significantly altered by feeding time in this study, night-fed bouts were shorter on average, therefore the increased number of bouts may be to compensate and to maximise energy savings. Other studies have found that torpor is primarily under circadian control, which can be influenced by food timing (Northeast et al., 2020; van der Vinne et al., 2018). As such, there may be competing forces contributing to torpor timing and characteristics (i.e., LD cycle, food timing) which may explain why some characteristics were altered but others were not.\n\nTorpor bout timing and duration between individuals and between days within individuals were found to be highly variable (Figure 1E). Moreover, our sample size was reduced due to it being deemed inappropriate to swap treatment groups as originally planned. Unfortunately, these factors limited our ability to compare between groups. We believe that with larger sample sizes, additional group differences would be found, and consider the current report preliminary.\n\nThe variation observed highlights the potential for torpor to confound experimental data due to differing torpor history. This may be an important consideration for behavioural tasks, as behavioural performance can be significantly affected by an animal’s experience (Milinski et al., 2021). One approach for minimising the effect of torpor on subsequent performance would be to control for feeding time by ensuring that food is provided at consistent times between days and for each animal. Often, time of feeding is not reported in the published literature; therefore, we recommend that time of feeding is clearly reported to facilitate replication of results and comparisons between studies.\n\nIt may be possible depending on the study to provide food at a time that will minimise the impact of torpor on subsequent experimental interventions. For example, food could be provided in the morning to shift torpor bouts to the dark phase, thus providing a window of opportunity to perform experimental manipulations. Where this is not possible, we recommend that mice are closely monitored for torpor bouts, for example using continuous thermal imaging, so that tasks are not being performed immediately following a torpor bout. Moreover, we recommend conducting tasks when torpor history, including number and depth of bouts, is comparable between individuals and days to minimise within- and between-individual variation.\n\nThese recommendations are relevant to all fields where food restriction is routinely used in mice and have the potential to refine food restriction procedures used in these fields. From a scientific perspective, we have identified that torpor is readily induced in laboratory mice in response to common food restriction protocols, and that time of feeding effects torpor characteristics. In relation to the 3Rs, remote measurement of Tskin via non-invasive thermal cameras will reduce stress in the animals. Moreover, continuous monitoring of Tskin may allow for early detection of unexpected or pathological hypothermia, enabling appropriate action to be initiated sooner. These recommendations will also contribute to improved data output with less variable data, resulting in reduced sample sizes required in future studies.\n\n\nAuthor contributions\n\nS.L.W, S.N.P, D.M.B and V.V.V designed experiments. S.L.W performed measurements. S.L.W and V.V.V performed analysis. S.L.W wrote the manuscript with input from all other authors.", "appendix": "Data availability\n\nFigshare: Induction of torpor in response to a common chronic food restriction paradigm and the effect of food timing on torpor characteristics.\n\n- Bodyweight measurements: https://doi.org/10.6084/m9.figshare.25722546 (Wilcox et al., 2024a).\n\n- Temperature recordings: https://doi.org/10.6084/m9.figshare.25722426 (Wilcox et al., 2024b).\n\nThis project contains the following underlying data:\n\n- Bodyweight.xlsx\n\n- 230920_1.dat\n\n- 230920_2.dat\n\n- 230920_3.dat\n\n- 230920_4.dat\n\n- 240920_1.dat\n\n- 240920_2.dat\n\n- 240920_3.dat\n\n- 240920_4.dat\n\n- 250920_1.dat\n\n- 250920_2.dat\n\n- 250920_3.dat\n\n- 250920_4.dat\n\n- 260920_1.dat\n\n- 260920_2.dat\n\n- 260920_3.dat\n\n- 260920_4.dat\n\n- 270920_1.dat\n\n- 270920_2.dat\n\n- 270920_3.dat\n\n- 270920_4.dat\n\n- 280920_1.dat\n\n- 280920_2.dat\n\n- 280920_3.dat\n\n- 280920_4.dat\n\n- 290920_1.dat\n\n- 290920_2.dat\n\n- 290920_3.dat\n\n- 290920_4.dat\n\n- 300920_1.dat\n\n- 300920_2.dat\n\n- 300920_3.dat\n\n- 300920_4.dat\n\n- 011020_1.dat\n\n- 011020_2.dat\n\n- 011020_3.dat\n\n- 011020_4.dat\n\n- 021020_1.dat\n\n- 021020_2.dat\n\n- 021020_3.dat\n\n- 021020_4.dat\n\n- 031020_1.dat\n\n- 031020_2.dat\n\n- 031020_3.dat\n\n- 031020_4.dat\n\n- 041020_1.dat\n\n- 041020_2.dat\n\n- 041020_3.dat\n\n- 041020_4.dat\n\n- 051020_1.dat\n\n- 051020_2.dat\n\n- 051020_3.dat\n\n- 051020_4.dat\n\n- 061020_1.dat\n\n- 061020_2.dat\n\n- 061020_3.dat\n\n- 061020_4.dat\n\n- 071020_1.dat\n\n- 071020_2.dat\n\n- 071020_3.dat\n\n- 071020_4.dat\n\n- 081020_1.dat\n\n- 081020_2.dat\n\n- 081020_3.dat\n\n- 081020_4.dat\n\n- 091020_1.dat\n\n- 091020_2.dat\n\n- 091020_3.dat\n\n- 091020_4.dat\n\n- 101020_1.dat\n\n- 101020_2.dat\n\n- 101020_3.dat\n\n- 101020_4.dat\n\n- 121020_1.dat\n\n- 121020_2.dat\n\n- 121020_3.dat\n\n- 121020_4.dat\n\n- 131020_1.dat\n\n- 131020_2.dat\n\n- 131020_3.dat\n\n- 131020_4.dat\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: ARRIVE author checklist: https://doi.org/10.6084/m9.figshare.26239967 (Wilcox, 2024c).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Laura E. McKillop and Vincent van der Vinne for assistance during the planning and running of experiments. We thank Sara Wells for her comments on this manuscript.\n\n\nReferences\n\nBrown JCL, Staples JF: Mitochondrial metabolism during fasting-induced daily torpor in mice. Bioenergetics. 2010; 1797: 476–486. PubMed Abstract \| Publisher Full Text\n\nFisher SP, Godinho SIH, Pothecary CA, et al.: Rapid assessment of sleep-wake behavior in mice.J. Biol. Rhythms.2012; 27(1): 48–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaskill BN, Rohr SA, Pajor EA, et al.: Working with what you’ve got: Changes in thermal preference and behavior in mice with or without nesting material. J. Therm. Biol. 2011; 36(3): 193–199. Publisher Full Text\n\nHuang YG, Flaherty SJ, Pothecary CA, et al.: The relationship between fasting-induced torpor, sleep, and wakefulness in laboratory mice. Sleep. 2021; 44(9): 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHudson JW, Scott IM: Daily torpor in the laboratory mouse, mus musculus var. albino. Physiol. Zool. 1979; 52(2): 205–218. Publisher Full Text\n\nLangford DJ, Bailey AL, Chanda ML, et al.: Coding of facial expressions of pain in the laboratory mouse. Nat. Methods. 2010; 7(6): 447–449. PubMed Abstract \| Publisher Full Text\n\nMelvin RG, Andrews MT: Torpor induction in mammals: Recent discoveries fueling new ideas. Trends Endocrinol. Metab. 2009; 20(10): 490–498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilinski L, Fisher SP, Cui N, et al.: Waking experience modulates sleep need in mice. BMC Biol. 2021; 19(1): 14–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNortheast RC, Vyazovskiy VV, Bechtold DA: Eat, sleep, repeat: the role of the circadian system in balancing sleep–wake control with metabolic need. Curr. Opin. Physio. 2020; 15: 183–191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOelkrug R, Heldmaier G, Meyer CW: Torpor patterns, arousal rates, and temporal organization of torpor entry in wildtype and UCP1-ablated mice. J. Comp. Physiol. B. 2011; 181(1): 137–145. PubMed Abstract \| Publisher Full Text\n\nSolymár M, Pétervári E, Balaskó M, et al.: The onset of daily torpor is regulated by the same low body mass in lean mice and in mice with diet-induced obesity. Temperature. 2015; 2(1): 129–134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwoap SJ, Gutilla MJ: Cardiovascular changes during daily torpor in the laboratory mouse. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 297(3): R769–R774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwoap SJ, Gutilla MJ, Liles LC, et al.: The Full Expression of Fasting-Induced Torpor Requires 3-Adrenergic Receptor Signaling. J. Neurosci. 2006; 26(1): 241–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTam SKE, Brown LA, Wilson TS, et al.: Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and shortterm memory.PNAS.2021; 118(39): e2101591118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Vinne V , Bingaman MJ, Weaver DR, et al.: Clocks and meals keep mice from being cool. J. Exp. Biol. 2018; 221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Vinne V , Pothecary CA, Wilcox SL, et al.: Continuous and non-invasive thermography of mouse skin accurately describes core body temperature patterns, but not absolute core temperature. Sci. Rep. 2020; 10(1): 20680–20689. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon der Ohe CG , Darian-Smith C, Garner CC, et al.: Ubiquitous and temperature-dependent neural plasticity in hibernators. J. Neurosci. 2006; 26(41): 10590–10598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilcox S, et al.: Bodyweight measurements. [Dataset]. figshare. 2024a. Publisher Full Text\n\nWilcox S, et al.: Body temperature recordings. [Dataset]. figshare. 2024b. Publisher Full Text\n\nWilcox S: ARRIVE author checklist: food timing. figshare. 2024c. Publisher Full Text\n\nWilcox S, et al.: Induction of torpor in response to a common chronic food restriction paradigm: implications for behavioural research using mice. [version 1; peer review: awaiting peer review]. F1000Res. 2024d. Publisher Full Text\n\nZhang Z, Reis FMCV, He Y, et al.: Estrogen-sensitive medial preoptic area neurons coordinate torpor in mice. Nat. Commun. 2020; 11(1): 6314–6378. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "317597", "date": "15 Oct 2024", "name": "Michael Ambler", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well thought out paper addressing an interesting question that is likely to be of interest to researchers in the field of torpor but also those using food restriction paradigms in other fields of study. The figures are clear, the methodology is sensible.\nWe note that the temperature recording files only appear to contain 4 minutes of data.\nMinor points:\nFigure 2 legend incorrectly references the figures. It describes E as representing change in the latency to the first torpor bout, when E is actually a graph about torpor bout duration. F-J all appear similarly mislabeled. The text body also refers to E as representing latency to torpor. “Additionally, no statistical difference in the relative timing of torpor over each 24h recording period, although night-fed mice entered torpor earlier on average (Morning: 14.2±0.4 ZTh, 95% CI [13.1–15.3]; Night: 12.2±0.9 ZTh, 95% CI [9.23–15.2]; p=0.0933, t(6)=1.99; Figure 2G).” – this line doesn’t quite make sense, are some words missing (e.g. ‘was observed’)? Last paragraph – “From a scientific perspective, we have identified that torpor is readily induced in laboratory mice in response to common food restriction protocols, and that time of feeding effects torpor characteristics” – should be affects not effects\n\nAdditional analysis to consider including:\nAnimal mass is given in a supplementary document. It is said the weights of the two groups were matched, but it would be helpful to see that detail in the text body (group average, sd). Starting body weight will affect an animal’s likelihood to enter torpor, as well as the latency to torpor entry. The figures only display 17 days of calorie restriction, but the weight charts go up to day 20. Were the animals ad-lib fed from day 17-20? Of not, why is this data not included? It would be interesting to add a comparison of total time in torpor. The authors have identified that night-fed mice opt for a strategy that includes more arousal from torpor. Does this result in less time spent torpid overall?\nPhrasing\nI believe the highlighted phrase in your abstract “Night-fed mice entered significantly more torpor bouts per day compared to morning-fed mice (Morning: 2.79±0.197 (mean ± SEM); Night: 4.79±0.533 (mean ± SEM); p=0.0125), but these bouts were shorter on average by ~1.5h” is slightly misleading. Greater clarity could be added to emphasize that the bouts were not significantly shorter, particularly given this later line in the text – “Although bout duration was not significantly altered by feeding time in this study”\n\nExplanation\nFor comparisons between groups, the last 6 days of recording are used as that is when bodyweight and temperature had “stabilized”. What was the criteria set for this stabilization? Looking at the bodyweight data, bodyweight seems to have stabilized by day 10. Is the last 6 days of recording day 11-17? Or day 14-20? In the linked paper(ref-1). bodyweight stabilized on day 8, so we are a little unclear on what / when defined stabilization for the comparison.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "331271", "date": "08 Nov 2024", "name": "Yoshifumi Yamaguchi", "expertise": [ "Reviewer Expertise Hibernation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes how food timing affects the propensity and timing of torpor in mice. Although the number of animals used is limited and the results should be considered preliminary as the authors themselves wrote, it sounds fine and provides useful information and caution to researchers who apply fasting or calorie-restriction protocols to mice in various fields, including metabolism, behavior, and neuroscience. I have made several comments that could be addressed.\nMajor comments: In Experimental design, “The morning-fed group and the night-fed group were housed in separate LTCs. The LD cycle for the night-fed group was shifted by one hour each day until a reverse LD cycle was achieved (lights on: 7 pm), taking a total of 12 days.” Was the LD cycle phase-advanced or -delayed to create a night-fed group? This information should be mentioned, because the efficiency of entrainment differs when the LD phase is advanced or delayed.\n\nIn addition, did you check the completion of entrainment of Tb rhythm to the LD-cycle before food restriction experiment in the morning-fed group? In other words, was the Tb rhythm before start of the food restriction the same between morning- and the night-fed groups? It could be evaluated by phase angle analysis described in the literature (Refinetti R, 1999). If entrainment was not completed in the morning-fed group, there is a possibility that the jet-lag condition may affect latency of torpor expression.\nFigure2 E–H: Does this correctly correspond to the main text?\n\nIn Discussion, “Although (torpor) bout duration was not significantly altered by feeding time in this study, night-fed bouts were shorter on average, therefore the increased number or bouts may be to compensate and to maximaise energy savings.” This can be verified by the dataset presented. Is the total duration of all torpor bouts over two weeks different or comparable between the night-fed and morning-fed groups? Alternatively, it can vary greatly among individuals. I understand that the small sample size may not allow such a comparison; however, data representation as a graph would be useful for readers.\n\nWhen food is given at a fixed time in the light phase, animals engage in food-anticipatory activity (Mistlberger RE, 1994; Vijaya Shankara J, 2022). Therefore, even though mice are fed at a fixed time in the light phase and thereby torpor no longer occur during the light phase, it should be considered that the food-anticipatory activity around the sampling time could affect the experimental results. This could be discussed.\nMinor comments: Figures 1B-D are the same as those shown in the report “Induction of torpor in response to a common chronic food restriction paradigm: implications for behavioural research using mice” by the same authors (Wilcox S, 2024). These figures do not seem essential to this study; it is better to omit them and instead cite the above paper.\nThe use of the words “morning feeding” and “night feeding” may be somewhat confusing. In nocturnal animals, “morning” (active) phase could be interpreted as early part of dark phase. It is better to add further explanation parallelly with the words “light-phase-fed” for morning feeding and “dark-phase-fed” for night feeding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-829
https://f1000research.com/articles/12-1602/v1	19 Dec 23	{ "type": "Research Article", "title": "Households with children under 5 years and older adults, and probability of expenditure on Food Away from Home (FAFH) in Peru in 2021: A Cross-Sectional Study", "authors": [ "Michelle Lozada-Urbano", "Aldo Alvarez-Risco", "Amalia Flores-Montero", "Juana Corzo-Ponte", "Franklin Huamán", "Joaquin Aguirre-Sosa", "Rosa Maria Benavente Ayquipa", "Oriana Rivera-Lozada", "Jaime A. Yáñez", "Aldo Alvarez-Risco", "Amalia Flores-Montero", "Juana Corzo-Ponte", "Franklin Huamán", "Joaquin Aguirre-Sosa", "Rosa Maria Benavente Ayquipa", "Oriana Rivera-Lozada", "Jaime A. Yáñez" ], "abstract": "For the CFAFH study, it is necessary to analyze the economic variable of families and the way it is distributed, since it allows a broader perspective of what a household invests in food consumption and how it is associated with health. The aim of this research was to determine the probability of expenditure on households that consume food away from home, with the presence of children under five years (Ch<5y) and households with the presence of older adults (HOA). A cross-sectional, descriptive, and quantitative study was carried out based on the grouping and analysis of data obtained from the Peruvian National Household Survey (ENAHO) 2021 database. After joining the database, the household type variables were created. Households with and without children under five years, and households with and without older adults were selected, as well as other variables such as the area of origin, gender of the head of the household, ethnicity, education level, marital status, age of the head of the household, occupation of the head of the household, and poverty. Descriptive statistics of the expenses made according to the variables studied and on the effect of each variable on the probability of purchase was obtained a through the application of the Logit model. These results warrant that it is necessary to generate awareness in the population about the food that is sold and consumed away from home for an appropriate selection of healthy options.", "keywords": [ "Food", "Households", "children", "Older adult", "ENAHO", "Food away from home", "expenditure" ], "content": "Introduction\n\nFood consumption patterns have fluctuated because of changes in the access to processed foods, presence of genetically modified organisms (GMO),1 urbanization and variations in income.2,3 Despite the preferential consumption of processed foods and the recent alarming increase in overweight and obesity,4–10 consumers are seeking functional foods because of their bioactive compound content,11–16 and with less pesticides,17 and that promote sustainability.18 On the other hand, there has been a reported increase of households that consume food away from home in part due to the increased offer of restaurants and food delivery services.3,19,20 Eating in restaurants has been linked with an increase in overweight children,21 and overweight and obese adults.22,23\n\nIt is estimated that in the world there are more than 41 million children under 5 years of age (Ch<5y) who are overweight or obese, situations that predispose them to health problems.24 In Peru, according to the demographic and family health survey (ENDES) of 2021, it was estimated that 9.6% of children under five years of age are overweight and obese.25 Likewise, 24.8% of older adults are overweight and 16.8% obese, figures that are higher than those of the previous decade.25 Overweight and obese children tend to continue to suffer from these conditions in adulthood and are more likely to suffer from non-communicable diseases such as diabetes, hypertension, heart disease, among others, at an increasingly younger age.26–30 This has been linked with a higher incidence of overweight and obese children and adults.21–23\n\nConsumption of food away from home (CFAFH) is a common eating habit and it has been reported that 42% of families in Peru do it and rank second in Latin America. As a result of globalization, fast food chains are a common option to purchase food away from home, which have been growing and improving the menu repertoire. In most cases, they offer food with low nutritional value, low in protein, vitamins, minerals, fiber and with high content of carbohydrates, saturated fats, cholesterol, showing a positive association with overweight,31–33 obesity,34–36 hypertension,37–39 diabetes,40–42 cardiovascular diseases43–45 and metabolic syndrome in adults46–48 and children.49–51 Furthermore, it has been reported that when dinner is consumed away from home, it is positively correlated with high body mass index (BMI).52,53\n\nThe amount and frequency of consumption of food away from home (CFAFH) depends on factors such as knowledge, beliefs, advertising and social acceptance of products, eating habits, and the purchasing power of households.54,55 Other factors such as the consumption patterns continuously fluctuate because of changes in food choice.1,2,18,56,57 On the other hand, there has been an increase in food delivery apps that have increased the consumption of food away from home (CFAFH)19,20 that are typically characterized for their poor nutritional quality.58–60\n\nChildren under 5 years old constitute a vulnerable population and are in a crucial stage of development, in which good nutrition and correct eating habits are imperative. In this sense, older adults are also affected by changes in diet and CFAFH because they need diet with adequate quantity and quality of nutrients. On the other hand, it is important to consider the socioeconomic level of the families, since the purchase of food away from home depends on the net economic income. This information will allow to observe the behavior of the families according to the variables studied.\n\nThe purpose of this study was to assess what are the characteristics of households that could have the probability of purchasing food away from home with the presence of children under five years of age (Ch<5y) and households with the presence of older adults (HOA). Our working hypothesis is that the expenditure of households with Ch<5y and households with the presence of older adults (HOA) will present at least one variable related to the probability of purchasing or not purchasing food away from home.\n\n\nLiterature review\n\nThe sustained growth of the Gross Domestic Product (GDP) in Peru between 1990 and 2018 was on average 4.90% per year, causing poverty to be reduced to 33.50% and the population in extreme poverty to 21.4%, and the Human Development Index (HDI) to improve from 0.61 to 0.75.61 Poverty is measured according to the people’s spending, so they will spend on goods or services that increase their well-being. The Poverty and Extreme Poverty Line has the cut-off point with the cost of the minimum basket that exclusively includes food, which was calculated for a person at 183 Peruvian Soles (19.28 US Dollars) per month and at 732 Peruvian Soles (193.29 US Dollars) per month for a family of 4 members in 2017.62\n\nPer capita spending on food in Peru in 2021 reached 40%, with 30.1% destined for food consumed at home (226 Peruvian Soles or 59.68 US Dollars) and 9.7% for food consumed away from home (73 Peruvian Soles or 19.28 US Dollars), thus implying that 40% of the expenses of Peruvians goes only to food. In the year 2022, the percentage has increased to 42.5% (INEI); however, the segments with less income, such as C, D and E spend approximately 45% of their salaries.63 It is important to consider that inflation is also related to consumption. In February 2022, Peru had a reported inflation of 6.15% that increased to 8.09% on May and it continues to increase.64 In households with lower incomes, there is a greater destination of money to food, being these households more sensitive to changes in the price of food.64\n\nIt has been reported that as the income increases there is an increase in the consumption of processed food with high amount of saturated fats,65,66 which correlates with the less time to prepare food at home because of increasing age and increasing responsibilities at the income increases.66,67 When there is less time to prepare food at home there is an increase in CFAH because of the quick access to food.56,68,69 During the pandemic, various countries provided financial support to the population, which increased income allowing for a higher food purchasing power.70 Food consumption patterns are directly linked with income based on the General Theory of Employment, Interest and Money.71 In this sense, the variability of local food prices is greater compared to foreign foods, which can mean that the population in cities far away from the capital or a major airport or from important roads do not cover their nutritional requirements. This has been observed in the city of Junin, that is located in the central highlands in Peru, and where 27.3% of the population do not cover their nutritional requirements with the food they have locally available.72 According to Corzo and Flores,73 in a study using the ENAHO database from 2017, households with children under five years of age and in households with older adults, the expense of consuming food away from home represents 8.29% and 6.83% of income, respectively. In another study published by our research group, it was observed that extremely poor households spend 4.14% or their income, non-extremely poor 6.45% and non-poor 7.20% based on the ENAHO database from 2019.3\n\n\nMethods\n\nThis research work took a free access database (secondary data). No names or documentation of the people are shown. This study had the approval of the Ethics Committee of the Norbert Wiener Private University (Registration No. 222-2020).\n\nA quantitative, cross-sectional, descriptive, and quantitative study was conducted using secondary source analysis from the National Household Survey (ENAHO) 2021 database, compiled by the National Institute of Statistics and Information (Instituto Nacional de Estadística e Informática - INEI).74 The survey was conducted at national level, in urban and rural areas in the 24 departments of Peru and in the Constitutional Province of Callao. The sampling was probabilistic of areas, stratified, multi-stage and independent in each department. The survey is freely accessible and is divided into five modules: education, employment and income, income from household work, characteristics of household members, and consumption of food away from home (CFAFH).\n\nThe population was composed of families in Peru, the sample was defined as the families in the urban and rural areas of Peru with the presence of children under five years of age (Ch<5y) and households with the presence of older adults (HOA). The total population corresponded to 34,584 households and after applying the expansion factor, we obtained 9,903,824 households. The processing of the National Household Survey (ENAHO) includes the use of an expansion factor, which is applied to each record and multiplied by all the data included in the record.74 The expansion factors are adjusted considering the population projections, including age and gender groups for each survey month and inference levels that are established with the sample design.\n\nThe inclusion criterion was that the household reported paying and consuming food away from home and met the characteristics of a household with the presence of children under five years of age and older adults. The independent variable was households with the presence of children under five years of age and older adults, and the sociodemographic characteristics of the head of household (area of origin, education, marital status, age of the head of household, gender, ethnicity, main occupation of the head of household, and poverty). The poverty variable was calculated in the ENAHO, and was divided into three groups: extremely poor, non-extremely poor, and non-poor. The dependent variable was expenditure on food consumed away from home.\n\nThe modules used for the development of this research correspond to modules 01 (characteristics of housing and household), 02 (characteristics of household members), 03 (education), 04 (health), 05 (employment) and 34 (module of calculated variables called summary) of the National Household Survey (ENAHO) 2021 database.74 The modules were separated; it was necessary to unite them through the STATA merge command. A first selection was made of households with children under five years of age and those without children. In the same way, households with older adults were selected, and those households without the presence of older adults in their composition. Household income was used to calculate how much was spent on food consumed away from home.\n\nThe average monthly expenditure on food consumption away from home was calculated and a first selection of households with children under five years of age and households without children was made. In the same way, households with older adults and households without the presence of older adults in their composition were selected. The variables used were the education level of the head of the household, the type of dwelling, the marital status of the head of the household, the age of the head of the household, the occupation of the head of the household, the average income of the household, the household income and the household poverty level calculated by the National Institute of Statistics and Information (INEI).\n\nThe household, presence of children under five years of age (lower end) and older adults (higher end) and area of origin, education, marital status, age of the head of household, gender, ethnicity, main occupation of the head of household, and poverty were used as variables. The main exposure variable of quantitative nature was the household that spent on food consumption away from home to conduct calculations considering a confidence level of 95%. Then, to construct the binary variable for this study, a dummy or dichotomous variable was used for the variable “expenditure on food consumed away from home - g05hd”, where 1 implied that the household purchased food away from home, and 0 when the household did not. It should be noted that this indicator was chosen for its ease of interpretation and modeling with other variables from the same survey. Households with and without children under five years of age, and households with and without older adults were selected.\n\nThe ENAHO 2021 data structure contains the weight variable, also known as expansion factor, in addition to the variable conglomerate or primary sampling unit and the sample stratum. These variables allowed us to define the sampling design of the survey, including corrections for finite populations and specify weights; for this, we used the svyset command in the STATA program. In this sense, the average expense was estimated using sample design framework, the command “svy: mean” and the option “over” (that measures the average expense by type of variable) for the detail at the category level of average spending. In these cases, the standard deviation and confidence intervals of each estimated category of spending was calculated. To know the variables that are part of the probability of purchase, first the means were estimated using the svy command, then the Logit command was used to obtain the probability of purchase of food consumed away from home. The primary sampling unit was the expenditure between the household with and without children under five years of age and older adults to obtain a population-level estimator based on the survey sample. The Stata Statistical Software, version 16 (StataCorp LLC) was used with statistical significance at p<0.05.\n\n\nResults\n\nThe data analyzed was obtained from the National Household Survey (ENAHO) 2021 version based on household records in Peru. These results include 9,903,824 families that consumed food away from home. Table 1 shows the average expenses per month, where households without children under 5 years of age spent on average 175.2 Peruvian Soles per month (approximately 46.26 US Dollars), households with children under 5 years of age spent 38.2 Peruvian Soles (approximately 10.09 US Dollars) more. In the case of households without any older adult, they spent 209.9 Peruvian Soles (approximately 55.43 US Dollars) on average per month, households with older adults spent 131.50 Peruvian Soles (approximately 34.72 US Dollars) less (Table 1).\n\nTable 2 according to the averaging comparison analysis, the result in the group of households with children under 5 years regarding the group of households without children indicates that the averages are statistically different, also for homes with older adults with respect to the group of households without Older adults indicates that averages are statistically different.\n\n_subpop_1: tipo_household_children = Household_without children <= 5 años\n\n_subpop_2: tipo_ household_children = Household with children <= 5 años\n\nHo: [GAFH]_subpop_1 - [GAFH]_subpop_2 = 0\n\n_subpop_1: tipo_household_older adults = Household without older adults\n\n_subpop_2: tipo_ household_older adults = Household with older adults\n\nHo: [GAFH]_subpop_1 - [GAFH]_subpop_2 = 0\n\nTable 3 shows the coefficients of the Logit model for households with Ch<5y and older adults. In households with children, the variables included in the expenditure model are the number of household members, when they increase by 1, there is a 4% higher probability of incurring in expenditure on FAFH. When the household is from a rural area, it has a 9% lower probability of incurring in expenditure on food consumed away from home. When the head of the household is a female, there is a 3% lower probability of incurring in expenditure on food consumed away from home. Likewise, if the head of the household is Aymara, there is a 12% probability of incurring in expenditure on FAFH. However, if the head of the household is a native person from the Amazon region, there is a 18% lower probability of incurring in expenditure on FAFH. If the head of the household is African Descendant, there is 11% lower probability of incurring in expenditure on FAFH. Head of households of mixed heritage have 8% lower probability of incurring in expenditure on FAFH. The educational level of the head of the household shows that there is a higher probability of incurring in expenditure on FAFH when the individual has: early education 26%, incomplete primary education 9%, complete primary education 10%, complete secondary education 5%, incomplete non-university higher education 5%. The head of the household in the 30-39 age range has 4% lower probability to spend on FAFH. Besides, when the head of the household is older than 60 years of age, there is also a 12% lower probability. When the head of the household is a dependent worker, there is a 3% lower probability of incurring in expenditure on FAFH. When the head of the household is non-extreme poor, there is a 15% probability of spending on FAFH. Non-poor heads of household have a 33% probability of spending on FAFH.\n\nIn households with older adults, the variables that are part of the expenditure model are the number of household members, when they increase by 1, there is a 5% higher probability of incurring in expenditure on FAFH. When the household is from a rural area, it has a 6% lower probability of incurring in expenditure on food consumed away from home. When the head of the household is a female, there is a 9% lower probability of incurring in expenditure on food consumed away from home. Likewise, if the head of the household is Aymara, there is a 14% probability of incurring in expenditure on FAFH. If the head of the household is African Descendant, there is 11% lower probability of incurring in expenditure on FAFH. If the head of the household is White, there is a 6% lower probability of spending on FAFH. Head of households of mixed heritage have 9% lower probability of incurring in expenditure on FAFH. The educational level of the head of the household shows that there is a higher probability of incurring in expenditure on FAFH when the individual has: incomplete primary education 5%, complete primary education 6%, complete secondary education 5%, incomplete non-university higher education 7%. When the head has incomplete university higher education, there is a 12% probability of spending on FAFH. When the head of the household is married, there is a 8% lower probability of incurring in expenditure on FAFH, and when the head is separated, there is a 8% probability of spending on FAFH. When the head of the household is a dependent worker, there is a 2% probability of incurring in expenditure on FAFH. When the head of the household is non-extreme poor, there is a 8% probability of spending on FAFH. Non-poor heads of household have a 23% probability of spending on FAFH.\n\n\nDiscussion\n\nThis paper assesses which variables are part of the probability of expenditure or non-expenditure on FAFH. In this study, it was found that households with Ch<5y spent an average of 213.4 Peruvian Soles (approximately 56.35 US Dollars) per month in food consumed away from home, which represents 4.94% of the income. In households with older adults (HOA), the expenditure on food consumed away from home was 131.5 Peruvian Soles (approximately 34.72 US Dollars), which represents 3.58% of income. This information in comparison with the available data in ENAHO 201775 indicates that the expenditure for Ch<5y, for the year 2021 decreased by 3.35%, while in the HOAs, the expenditure in the year 2017 was 6.83%, which shows a decrease for the year 2021 by 3.25%.\n\nQueiroz and Coelho76 found that hiring domestic help decreases expenditure on FAFH, especially on lunches and dinners, and that the presence of children in the household decreases consumption of most categories of FAFH. This alternative against FAFH is possible in Peru in families in socioeconomic levels A and B mainly due to the associated salary cost for the hired person. In this study, the definition of the variables non-extreme poor and non-poor favored the probability of expenditure on FAFH. In contrast to what was found in this study, Aguila-Lopez and Kuhar77 reported that in Mexico the household share of food expenditure away from home decreased in all income deciles and in all regions, but the impact on household participation is far from uniform. Furthermore, they highlight that an increase or decrease in income will lead to a corresponding change in expenditure on FAFH, but the change will be less than proportional.77\n\nCrespo et al.68 reported that although the frequency of FAFH was similar across all levels of food security (FS), adults with high FS spent more dollars ($213.60) and a greater proportion (29.4%) of their food budget on FAFH compared to adults with marginal, low and very low FS ($133.00, $116.20, $103.30 and 21.4%, 19.7% and 20.0%, respectively). They also reported that the prevalence of obesity was higher in adults with a low (42.9%) and very low (41.5%) FS, and lower in adults with a high FS (33.7%).68 These are similar to those reported since expenditure on FAFH is related to family income.\n\nOn average for Peru in 2021, the average expenditure on food consumed away from home reaches between 4.94 and 3.58% for households with Ch<5y and HOAs, respectively. In that sense, although a reduction in four years is notorious, there is continuity in the consumption of food bought on the street, without regulation; this could be an indication that in these homes children and older adults are not being fed with healthy food. Reuter found a positive correlation between higher rates of overweight and obesity and higher consumption of fast food in restaurants, with children being the most affected.23 In other studies in Peru on food consumed in the home, Lozada-Urbano et al. found that the highest expenditure was made by households defined as composite.3 However, although in this study the same classification was not made in households with children where the head of household is widowed who spends more, and in households with older adults more is spent when the head of household is a cohabitant.3 In contrast to these findings, the separated marital status in households with older adults was a factor that favors the probability of expenditure on FAFH. Likewise, Jia et al. also found in their study with children under 18 years of age, a positive association between eating away from home and overweight and obesity.60 Some other variables that favor the probability of expenditure and therefore consumption in households with children under five years of age are the increase in the number of household members, being native Aymara or other native people, having early education, complete primary education, being a dependent worker.\n\nRegarding the occupation of the head of household, in households with children and adults, we observed that the head of household with dependent work is the one who spends the most on food consumed away from home and households with children spend more (36.8 Peruvian Soles, approximately 9.72 US Dollars) than households with older adults. Similarly, heads of households with dependent work was a factor that favored the probability of expenditure on FAFH for both households with children and adults. It has been reported that with every 10% increase in average per capita income, there is a 3.5% increase in the proportion of food consumed away from home.78 In order to achieve better nutrition at home and avoid spending on eating out requires action on the part of parents but as reported by Eck et al.,79 lack of time and busy schedules are the most common barriers. Also, a major obstacle to ensure that children eat healthy is that parents are not present to monitor children’s intake.79 Importantly, children were also surveyed and reported to understand that frequent eating away from home resulted in less healthy behaviors.79 This last data is of great value to generate studies that address the children’s point of view for these eating patterns and strategies focused on adults, adolescents and children can be generated.\n\nThis study does not allow us to know the name of the foods or preparations consumed by the respondents and therefore we cannot calculate the calories consumed. It is also not known whether the food purchased was shared by all the members in the household. The differences shown in other studies show that those with a high socioeconomic level spend more and purchase healthy foods, while the poorest households purchase unhealthy foods.\n\nFor society, it is important to create and maintain awareness of the quality of the food consumed away from home due to the exposure to trans fats, sodium and sugar that leads to chronic diseases. For the government, it is critical to create policies that support educating the population on nutrition and healthy eating issues considering sociodemographic characteristics. A good and timely education should be aimed at those who consume and those who prepare food. A consumption of healthy foods can help in preventing diseases that would make investment in health more expensive and would alter the quality of life. For the university, nutrition schools can invest in research on nutritional health issues, and this study can help to propose new state policies. Nutrition professionals can work on practical proposals for the development of counseling and training spaces on consumption of food away from home issues in children under 5 years of age and older adults. Menu labeling can reduce the energy content of foods consumed away from home and make it easier for consumers to choose healthier items.\n\n\nConclusions\n\nHouseholds with children under 5 years of age spend more on food consumed away from home than households with older adults. In households with children, the variables that favor the probability of expenditure on FAFH are: the number of household members, being Aymara or from another native people, having early education, complete primary education, being a dependent worker, being non-extreme poor, and being non-poor. Variables that decrease the probability of expenditure are: when the head of the household is from a rural area, is native from the Amazon region, is African descendant, has mixed heritage, is between 30 and 39 years of age, and is over 60 years of age.\n\nIn households with older adults, the variables that favor the probability of expenditure on FAFH are: the number of household members, being Aymara or from another native people, having incomplete primary education, complete primary education, complete secondary education, incomplete higher university education, being a dependent worker, being separated, being non-extreme poor, and being non-poor. Variables that decrease the probability of expenditure are: when the head of the household is from a rural area, is native from the Amazon region, is African descendant, is White, has mixed heritage, is native from an indigenous people, is married.\n\nFamilies with children and older adults need to know which are the foods and meals that favor the nutrition of these age groups, to ensure good habits and good nutrition in them. Likewise, it is necessary to have a control of the quality of food in restaurants and street vendors, to offer healthy consumption options.", "appendix": "Data availability\n\nOlder adults and children (Food consumed outside the home) https://doi.org/10.6084/m9.figshare.23935221.v1. 80\n\nThis project contains the following underlying data:\n\n• DATA_niño&adultomayor_FIGSHARE 11 08.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nDelgado-Zegarra J, Alvarez-Risco A, Cárdenas C, et al.: Labeling of Genetically Modified (GM) Foods in Peru: Current Dogma and Insights of the Regulatory and Legal Statutes. Int. J. Food Sci. 2022; 2022: 3489712–3489785. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhai FY, Du SF, Wang ZH, et al.: Dynamics of the Chinese diet and the role of urbanicity, 1991–2011. Obes. Rev. 2014; 15: 16–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLozada-Urbano M, Huamán F, Xirinachs Y, et al.: Poverty, Household Structure and Consumption of Foods Away from Home in Peru in 2019: A Cross-Sectional Study. Foods. 2022; 11: 2547. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeramat SA, Alam K, Al-Hanawi MK, et al.: Trends in the prevalence of adult overweight and obesity in Australia, and its association with geographic remoteness. Sci. Rep. 2021; 11: 11320. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpinelli A, Buoncristiano M, Nardone P, et al.: Thinness, overweight, and obesity in 6- to 9-year-old children from 36 countries: The World Health Organization European Childhood Obesity Surveillance Initiative—COSI 2015–2017. Obes. Rev. 2021; 22: e13214. PubMed Abstract \| Publisher Full Text\n\nAgustina R, Meilianawati, Fenny, et al.: Psychosocial, eating behavior, and lifestyle factors influencing overweight and obesity in adolescents. Food Nutr. Bull. 2021; 42: S72–S91. PubMed Abstract \| Publisher Full Text\n\nHecker J, Freijer K, Hiligsmann M, et al.: Burden of disease study of overweight and obesity; the societal impact in terms of cost-of-illness and health-related quality of life. BMC Public Health. 2022; 22: 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChew HSJ, Gao Y, Shabbir A, et al.: Personal motivation, self-regulation barriers and strategies for weight loss in people with overweight and obesity: a thematic framework analysis. Public Health Nutr. 2022; 25: 2426–2435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoś K, Rychlik E, Woźniak A, et al.: Prevalence and Sociodemographic Factors Associated with Overweight and Obesity among Adults in Poland: A 2019/2020 Nationwide Cross-Sectional Survey. Int. J. Environ. Res. Public Health. 2022; 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZervos K, Koletsi M, Mantzios M, et al.: An Eight-Week Mindful Eating Program Applied in a Mediterranean Population With Overweight or Obesity: The EATT Intervention Study. Psychol. Rep. 2021; 125: 1011–1040. PubMed Abstract \| Publisher Full Text\n\nBermudez-Aguirre D, Yáñez J, Dunne C, et al.: Study of strawberry flavored milk under pulsed electric field processing. Food Res. Int. 2010; 43: 2201–2207. Publisher Full Text\n\nYáñez JA, Miranda ND, Remsberg CM, et al.: Stereospecific high-performance liquid chromatographic analysis of eriodictyol in urine. J. Pharm. Biomed. Anal. 2007; 43: 255–262. PubMed Abstract \| Publisher Full Text\n\nVega-Villa KR, Remsberg CM, Ohgami Y, et al.: Stereospecific high-performance liquid chromatography of taxifolin, applications in pharmacokinetics, and determination in tu fu ling (Rhizoma smilacis glabrae) and apple (Malus x domestica). Biomed. Chromatogr. 2009; 23: 638–646. PubMed Abstract \| Publisher Full Text\n\nRamos-Escudero F, Santos-Buelga C, Pérez-Alonso JJ, et al.: HPLC-DAD-ESI/MS identification of anthocyanins in Dioscorea trifida L. yam tubers (purple sachapapa). Eur. Food Res. Technol. 2010; 230: 745–752. Publisher Full Text\n\nRoupe KA, Helms GL, Halls SC, et al.: Preparative enzymatic synthesis and HPLC analysis of rhapontigenin: applications to metabolism, pharmacokinetics and anti-cancer studies. J. Pharm. Pharm. Sci. 2005; 8: 374–386. PubMed Abstract\n\nYáñez JA, Remsberg CM, Takemoto JK, et al.: Polyphenols and Flavonoids: An Overview. Flavonoid Pharmacokinetics: Methods of Analysis, Preclinical and Clinical Pharmacokinetics, Safety, and Toxicology. Davies NM, Yáñez JA, eitors. Hoboken, NJ USA: John Wiley & Sons; 2012; pp. 1–69.\n\nDelgado-Zegarra J, Alvarez-Risco A, Yáñez JA: Indiscriminate use of pesticides and lack of sanitary control in the domestic market in PeruUso indiscriminado de pesticidas e falta de controle sanitário do mercado interno no Peru. Rev. Panam. Salud Publica. 2018; 42: e3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvarez-Risco A, Delgado-Zegarra J, Yáñez JA, et al.: Predation Risk by Gastronomic Boom - Case Peru. J. Landsc. Ecol. 2018; 11: 100–103. Publisher Full Text\n\nPenney TL, Burgoine T, Monsivais P: Relative Density of Away from Home Food Establishments and Food Spend for 24,047 Households in England: A Cross-Sectional Study. Int. J. Environ. Res. Public Health. 2018; 15: 2821. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTian X, Zhong L, von Cramon-Taubadel S , et al.: Restaurants in the Neighborhood, Eating Away from Home and BMI in China. PLoS One. 2016; 11: e0167721. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMachado-Rodrigues AM, Gama A, Mourão I, et al.: Eating away from home: a risk factor for overweight in children. Eur. J. Clin. Nutr. 2018; 72: 1724–1727. PubMed Abstract \| Publisher Full Text\n\nLachat C, Nago E, Verstraeten R, et al.: Eating out of home and its association with dietary intake: a systematic review of the evidence. Obes. Rev. 2012; 13: 329–346. PubMed Abstract \| Publisher Full Text\n\nKim TH, Lee E-K, Han E: Food away from home and body mass outcomes: Taking heterogeneity into account enhances quality of results. Nutrition (Burbank, Los Angeles County, Calif.). 2014; 30: 1015–1021. PubMed Abstract \| Publisher Full Text\n\nAlshahrani A, Shuweihdi F, Swift J, et al.: Underestimation of overweight weight status in children and adolescents aged 0-19 years: A systematic review and meta-analysis. Obes. Sci. Pract. 2021; 7: 760–796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nINEI: Encuesta Demográfica y de Salud Familiar, 2021 [Demographic and Family Health Survey, 2021].2021.\n\nMakni E, Elloumi A, Ben Brahim M, et al.: Six-minute walk distance equation in children and adolescents with obesity. Acta Paediatr. 2020; 109: 2729–2737. PubMed Abstract \| Publisher Full Text\n\nSanyaolu A, Okorie C, Qi X, et al.: Childhood and Adolescent Obesity in the United States: A Public Health Concern. Glob. Pediatr. Health. 2019; 6: 2333794X19891305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaprio S, Santoro N, Weiss R: Childhood obesity and the associated rise in cardiometabolic complications. Nat. Metab. 2020; 2: 223–232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSafaei M, Sundararajan EA, Driss M, et al.: A systematic literature review on obesity: Understanding the causes & consequences of obesity and reviewing various machine learning approaches used to predict obesity. Comput. Biol. Med. 2021; 136: 104754. Publisher Full Text\n\nKnebusch V, Williams J, Yordi Aguirre I, et al.: Effects of the coronavirus disease 2019 pandemic and the policy response on childhood obesity risk factors: Gender and sex differences and recommendations for research. Obes. Rev. 2021; 22: e13222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanhada SL, Luft VC, Giatti L, et al.: Ultra-processed foods, incident overweight and obesity, and longitudinal changes in weight and waist circumference: the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). Public Health Nutr. 2020; 23: 1076–1086. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAskari M, Heshmati J, Shahinfar H, et al.: Ultra-processed food and the risk of overweight and obesity: a systematic review and meta-analysis of observational studies. Int. J. Obes. 2020; 44: 2080–2091. PubMed Abstract \| Publisher Full Text\n\nSartorelli DS, Crivellenti LC, Baroni NF, et al.: Effectiveness of a minimally processed food-based nutritional counselling intervention on weight gain in overweight pregnant women: a randomized controlled trial. Eur. J. Nutr. 2022; 62: 443–454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPassos CM, Maia EG, Levy RB, et al.: Association between the price of ultra-processed foods and obesity in Brazil. Nutr. Metab. Cardiovasc. Dis. 2020; 30: 589–598. Publisher Full Text\n\nNardocci M, Leclerc B-S, Louzada M-L, et al.: Consumption of ultra-processed foods and obesity in Canada. Can. J. Public Health. 2019; 110: 4–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarb AA, Shechter A, Koch PA, et al.: Ultra-processed foods and the development of obesity in adults. Eur. J. Clin. Nutr. 2022; 77: 619–627. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarbosa SS, Sousa LC, de Oliveira Silva DF , et al.: A Systematic Review on Processed/Ultra-Processed Foods and Arterial Hypertension in Adults and Older People. Nutrients. 2022; 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang M, Du X, Huang W, et al.: Ultra-Processed Foods Consumption Increases the Risk of Hypertension in Adults: A Systematic Review and Meta-Analysis. Am. J. Hypertens. 2022; 35: 892–901. PubMed Abstract \| Publisher Full Text\n\nScaranni POS, Cardoso LO, Chor D, et al.: Ultra-processed foods, changes in blood pressure and incidence of hypertension: the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). Public Health Nutr. 2021; 24: 3352–3360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeone A, Martínez-González MÁ, Craig W, et al.: Pre-Gestational Consumption of Ultra-Processed Foods and Risk of Gestational Diabetes in a Mediterranean Cohort. The SUN Project. Nutrients. 2021; 13. Publisher Full Text\n\nLlavero-Valero M, Escalada-San Martín J, Martínez-González MA, et al.: Ultra-processed foods and type-2 diabetes risk in the SUN project: A prospective cohort study. Clin. Nutr. 2021; 40: 2817–2824. PubMed Abstract \| Publisher Full Text\n\nNardocci M, Polsky JY, Moubarac J-C: Consumption of ultra-processed foods is associated with obesity, diabetes and hypertension in Canadian adults. Can. J. Public Health. 2021; 112: 421–429. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuul F, Vaidean G, Lin Y, et al.: Ultra-Processed Foods and Incident Cardiovascular Disease in the Framingham Offspring Study. J. Am. Coll. Cardiol. 2021; 77: 1520–1531. PubMed Abstract \| Publisher Full Text\n\nBonaccio M, Costanzo S, Di Castelnuovo A, et al.: Ultra-processed food intake and all-cause and cause-specific mortality in individuals with cardiovascular disease: the Moli-sani Study. Eur. Heart J. 2022; 43: 213–224. PubMed Abstract \| Publisher Full Text\n\nMatos RA, Adams M, Sabaté J: Review: The Consumption of Ultra-Processed Foods and Non-communicable Diseases in Latin America. Front. Nutr. 2021; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrimarco A, Landry MJ, Gardner CD: Ultra-processed Foods, Weight Gain, and Co-morbidity Risk. Curr. Obes. Rep. 2022; 11: 80–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIvancovsky-Wajcman D, Fliss-Isakov N, Webb M, et al.: Ultra-processed food is associated with features of metabolic syndrome and non-alcoholic fatty liver disease. Liver Int. 2021; 41: 2635–2645. PubMed Abstract \| Publisher Full Text\n\nJuul F, Deierlein AL, Vaidean G, et al.: Ultra-processed Foods and Cardiometabolic Health Outcomes: from Evidence to Practice. Curr. Atheroscler. Rep. 2022; 24: 849–860. PubMed Abstract \| Publisher Full Text\n\nFilgueiras AR, Pires de Almeida VB, Koch Nogueira PC, et al.: Exploring the consumption of ultra-processed foods and its association with food addiction in overweight children. Appetite. 2019; 135: 137–145. PubMed Abstract \| Publisher Full Text\n\nSirkka O, Fleischmann M, Abrahamse-Berkeveld M, et al.: Dietary Patterns in Early Childhood and the Risk of Childhood Overweight: The GECKO Drenthe Birth Cohort. Nutrients. 2021; 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoubiap JJ, Nansseu JR, Lontchi-Yimagou E, et al.: Global, regional, and country estimates of metabolic syndrome burden in children and adolescents in 2020: a systematic review and modelling analysis. Lancet Child Adolesc. Health. 2022; 6: 158–170. PubMed Abstract \| Publisher Full Text\n\nSeguin RA, Aggarwal A, Vermeylen F, et al.: Consumption Frequency of Foods Away from Home Linked with Higher Body Mass Index and Lower Fruit and Vegetable Intake among Adults: A Cross-Sectional Study. J. Environ. Public Health. 2016; 2016: 3074212–3074241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalleh R, Ganapathy SS, Ibrahim Wong N, et al.: Is socio-demographic status, body mass index, and consumption of food away from home associated with high sodium intake among adults in Malaysia?: findings from the Malaysian Community Salt Survey (MyCoSS). J. Health Popul. Nutr. 2021; 40: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinkley JK: The Effect of Demographic, Economic, and Nutrition Factors on the Frequency of Food Away from Home. J. Consum. Aff. 2006; 40: 372–391. Publisher Full Text\n\nLandais E, Miotto-Plessis M, Bene C, et al.: Consumption of food away from home in low- and middle-income countries: a systematic scoping review. Nutr. Rev. 2022; 81: 727–754. Publisher Full Text\n\nGarcía Arancibia R, Depetris-Guiguet E, Rossini G: Consumo de alimentos fuera del hogar en Argentina. Relevancia de la composición demográfica y tipología de los hogares. Población y Salud en Mesoamérica. 2013; 10: 1–19.\n\nDelgado-Zegarra J, Alvarez-Risco A, Yáñez JA: Indiscriminate use of pesticides and lack of sanitary control in the domestic market in Peru. Revista Panamericana de Salud Publica/Pan American Journal of Public Health. 2018; 42: e3.\n\nAbegunde DO, Mathers CD, Adam T, et al.: The burden and costs of chronic diseases in low-income and middle-income countries. Lancet. 2007; 370: 1929–1938. Publisher Full Text\n\nVillacis C, Zazpe I, Santiago S, et al.: Frecuencia de comidas fuera de casa y calidad de hidratos de carbono y de grasas en el Proyecto SUN. Nutr. Hosp. 2015; 31: 466–474.\n\nJia X, Liu J, Chen B, et al.: Differences in nutrient and energy contents of commonly consumed dishes prepared in restaurants v. at home in Hunan Province, China. Public Health Nutr. 2018; 21: 1307–1318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbelardo Hurtado V, Wadson P: Crecimiento económico, pobreza y desarrollo humano en el Perú. Revista Científica Pakamuros. 2019; 7: 68–79. Publisher Full Text\n\nParodi C: ¿Cómo se mide la pobreza? (accessed on 28 December 2022). Reference Source\n\nA pesar de la inflación, el 40% del gaasto de los hogares peruanos se destina a alimentos. (accessed on 20 December 2022). Reference Source\n\nEl González D : 40% del gasto de los hogares peruanos se destina a alimentos. (accessed on 20 December 2022). Reference Source\n\nLópez de Blanco M, Carmona A: La transición alimentaria y nutricional: Un reto en el siglo XXI. Anales Venezolanos de Nutrición. 2005; 18: 90–104.\n\nVenn D, Dixon J, Banwell C, et al.: Social determinants of household food expenditure in Australia: the role of education, income, geography and time. Public Health Nutr. 2018; 21: 902–911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaillie LS, Afeiche MC, Eldridge AL, et al.: The contribution of at-home and away-from-home food to dietary intake among 2–13-year-old Mexican children. Public Health Nutr. 2017; 20: 2559–2568. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrespo-Bellido MS, Grutzmacher SK, Takata Y, et al.: The Association Between Food-Away-From-Home Frequency and a Higher BMI Varies by Food Security Status in US Adults. J. Nutr. 2021; 151: 387–394. PubMed Abstract \| Publisher Full Text\n\nSmith LP, Ng SW, Popkin BM: Trends in US home food preparation and consumption: analysis of national nutrition surveys and time use studies from 1965–1966 to 2007–2008. Nutr. J. 2013; 12: 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhammad A, Seale JL, Meade B, et al.: International Evidence on Food Consumption Patterns: An Update Using 2005 International Comparison Program Data. U.S. Department of Agriculture, Economic Research Service; 2011.\n\nJean De L: A Note on the “General Theory of Employment, Interest and Money”. J. Post Keynes. Econ. 1979; 1: 6–15. Publisher Full Text\n\nRosales G, Mercado W: Efecto de los cambios en el precio de los alimentos sobre el consumo de la quinua y la seguridad alimentaria rural en el Perú. Sci. Agropecu. 2020; 11: 83–93. Publisher Full Text\n\nCorzo Ponte JM, Flores Montero AE: Gasto en el consumo de alimentos fuera del hogar, y cuánto representa del ingreso familiar, en hogares con presencia de niños menores de cinco años y hogares con presencia de adultos mayores-2017.2020.\n\nINEI: Encuesta Nacional de Hogares ENAHO [National Household Survey ENAHO]. Peru: INEI; 2021.\n\nINEI: Encuesta Nacional de Hogares ENAHO [National Household Survey ENAHO]. Peru: INEI; 2017.\n\nQueiroz P, Coelho AB: Food away from home in Brazil: the role of sociodemographic factors and family structure. Int. J. Soc. Econ. 2019; 46: 503–522. Publisher Full Text\n\nAguilar-Lopez A, Kuhar A: Food-Away-from-Home Expenditure in Mexico during the COVID-19 Pandemic: A Micro-Econometric Analysis. Agriculture. 2022; 12. Publisher Full Text\n\nCunha DB, Bezerra IN, Pereira RA, et al.: At-home and away-from-home dietary patterns and BMI z-scores in Brazilian adolescents. Appetite. 2018; 120: 374–380. PubMed Abstract \| Publisher Full Text\n\nEck KM, Delaney C, Olfert MD, et al.: Parents’ and kids’ eating away from home cognitions. Br. Food J. 2019; 121: 1168–1182. Publisher Full Text\n\nLozada-urbano M, Huamán F: Older adults and children (Food consumed outside the home). Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "249950", "date": "21 Mar 2024", "name": "Olutosin Ademola Otekunrin", "expertise": [ "Reviewer Expertise Agricultural Economics", "Food (In)security", "Zero Hunger", "Agricultural commercialization", "Farm Management", "Food Economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFrom the abstract, it is important that authors give the full meaning of the acronym CFAFH at first mention. Add the sample size using ENAHO 2021 database. It was quite obvious that authors did not present any of the findings in this section. In the introduction, having 23 references in a single paragraph is rather unwarranted. Kindly reduce it. However, on a general note, I am not comfortable with the rationale for referencing in this study. In the introduction, a total of 73 refs were included. Please, do the needful. You are expected to present both implicit and explicit specification for the regression model used in this study. Kindly show the equations for for this model. List all the variables of interest, both dependent(s) and explanatory variables. From the discussion, you did not discuss your results of your Logit model in Table 3. Use asterisks to denote the significant levels in your model result 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? 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": "11501", "date": "24 Jul 2024", "name": "Michelle Lozada-Urbano", "role": "Author Response", "response": "Dear reviewer, we appreciate all your suggestions, all changes have been made and are shown in the new version of the document" } ] } ]	1	https://f1000research.com/articles/12-1602
https://f1000research.com/articles/13-827/v1	24 Jul 24	{ "type": "Research Article", "title": "Adjusting for covariates representing potential confounders, mediators, or competing predictors in the presence of measurement error: Dispelling a potential misapprehension and insights for optimal study design with nutritional epidemiology examples", "authors": [ "Roger S. Zoh", "Diana M. Thomas", "Carmen D. Tekwe", "Xiaoxin Yu", "Colby J. Vorland", "Nikhil V. Dhurandhar", "David M. Klurfeld", "David B. Allison", "Diana M. Thomas", "Carmen D. Tekwe", "Xiaoxin Yu", "Colby J. Vorland", "Nikhil V. Dhurandhar", "David M. Klurfeld" ], "abstract": "Background Variables such as dietary intake are measured with error yet frequently used in observational epidemiology. Although this limitation is sometimes noted, these variables are still often modeled as covariates without formal correction or sincere dialogue about measurement unreliability potentially weakening the validity of statistical conclusions. Further, larger sample sizes increase power (bias) to detect spurious correlations. Counterintuitively, recent work suggested a non-monotonic relationship between confounder unreliability and how much controlling for the confounder reduces (or induces) bias when testing for an exposure-outcome association. If true, such non-monotonicity would be especially concerning for applications such as nutrition, where measurement reliability varies substantially, and large sample sizes are common.\n\nMethods We offer a detailed derivations of the square partial correlation between the outcome and exposure, controlling for the confounder. In our derivation, the measurement reliabilities of exposures and confounders are not arbitrarily constrained to be equal. Further, our theoretical results are investigated using simulations.\n\nResults Reassuringly, these derivations and simulations show that the counterintuitive non-monotonicity relationship between confounder unreliability and how much controlling for the confounder reduces (or induces) bias when testing for an exposure-outcome association is an artifact of the arbitrary constraint which forces the measurement reliabilities of exposures and confounders to be equal, which that does not always hold.\n\nConclusions The profound and manifold effects of measurement error on estimation and statistical conclusion validity in realistic scenarios indicate that merely mentioning measurement error as a limitation and then dispensing with it is not an adequate response. We also explore questions for optimal study design subject to resource constraints when considering reliability of exposures, covariates, and outcomes.", "keywords": [ "Measurement Error", "Bias", "Measurement reliability", "Type I Error", "Covariate Adjustment" ], "content": "Introduction\n\nThe challenges of measuring dietary intake and other variables in observational epidemiology in nutrition and obesity research have been widely discussed.1–3 Data on nutrient or energy intake inform prevention and treatment guidance and policies for many health conditions. Fidelity of data to true nutrient and energy intake is essentially related to the ability to draw valid scientific conclusions about nutritional effects. It is not easy to obtain food intake information, however, particularly from a large number of individuals as is done for the highly reputable and influential National Health and Nutrition Examination Survey (NHANES). NHANES obtains nutrient and energy intake data through self-reported dietary recall questionnaires. This approach involves individuals reporting food and drinks consumed in the past 24 hours followed by detailed questions to accurately gauge the amounts of nutrients and energy consumed. Another such subjective approach that is popular is the food-frequency questionnaire, which asks individuals about the frequency of consumption of various foods.\n\nUnderstandably, such information related to nutrient and energy intake is influenced by intentional or unintentional misreporting, which substantially impacts accuracy. For example, a study pooled data from five large studies that had examined the validity of food-frequency questionnaires and 24-h dietary recalls against recovery biomarkers. Across this diverse sample of Americans, subjective estimates of energy intake explained less than 10% of the variance in true energy intake.1 It appears that the self-reported measures systematically underestimate energy intake by hundreds of kcal per day, and the NHANES surveys may underreport energy intake by as much as 800 kcal per day.2,3\n\nAccuracy of data should be a concern for any researcher, and this level of inaccuracy should be disconcerting. However, it is a common practice to acknowledge the limitations of such measurements and to then carry on with the research as though it has at least some validity despite these limitations. Some common practices to dismiss or minimize concerns around lack of accuracy of data include rationalization that self-reported data about energy or nutrient intake have good reproducibility, or that they could be adjusted by a common factor.4 There are serious counterarguments against such rationalizations. Settling for reproducibility of data instead of accuracy is like using a mismarked measuring tape. One can reproduce the result, but it is still inaccurate. Also, dietary underreporting varies with the type of food consumed, gender, age, smoking habits, social class, education level, dietary restraints, body mass index (BMI) of the respondents, and other life stage factors,5 which makes it implausible that one could adequately correct a given dataset by a single factor and arithmetic operation.\n\nConsidering the serious implications of using decidedly inaccurate information, some investigators have raised concerns that some limitations are so severe that we are better off not conducting certain research or not using certain methods at all until more accurate methods become available. For example, Lear wrote “One may wonder if we should stop nutritional research altogether until we can get it right”.4 While this may be an extreme rhetorical position, Dhurandhar et al.5 noted that in some cases, measurement properties are so bad that it is better to not perform the research at all than to use the poor measurement instruments (even if the instruments are the best available for a particular context). Putting it succinctly, on some occasions, something is not better than nothing.\n\nRecently, we became aware of a particular finding regarding the effects of controlling for a confounding variable that is measured with error. Westfall and Yarkoni6 showed results that seemed to imply that there is not necessarily a monotonic relationship between (a) the degree of measurement error in a confounding variable and (b) the extent to which controlling for the measurement of the confounding variable reduces bias in testing for an exposure association with an outcome. Rather, the degree of bias in testing appears to increase as one moves from a completely unreliable measure of the confounder to a partially reliable measure and then decreases again as one approaches a perfectly reliable measure.\n\nNote that in frequentist significance testing (the type of testing generally used in this journal), a test may be said to be biased when the sampling distribution of P values from applying the test is not uniform on the interval [0,1] under the null hypothesis.7 The main concern is that the “size of the test” is inflated such that, in probability, too many small P values are generated, leading to too many type I errors.\n\nAnother key point from the article by Westfall and Yarkoni6 that appears counterintuitive is that the larger the sample size, the higher the error rate. This is critical in nutritional epidemiology because greater importance is regularly given to the largest cohort studies when in fact the largest studies may be the most susceptible to biasing in significance testing both because they have more power to detect all effects, including spurious ones, and because large scale studies may be less well able to measure variables well.8\n\nAlthough we were able to identify some articles in the nutrition and obesity literature that cited the Westfall and Yarkoni article,9–17 none of them addressed the issue of modest reliability potentially being worse than poorer reliability. To the best of our ability to discern, this issue has not been recognized in the nutrition and obesity literature. Yet, this methodologic issue in which intermediary measurement reliability may be worse than either very low or very high reliability seems especially concerning for the nutritional epidemiology field, where even aspects of dietary intake that can be measured with some degree of reliability and validity often have very modest degrees of reliability or validity.18 Thus, this non-monotonicity problem may be a particular concern, and if true, might suggest the startling conclusion that in nutrition epidemiology it is better to not measure some covariates at all than to include them in the study, if they have only modest reliability.\n\nHere we provide an explanation of the phenomenon in simple scenarios so that researchers, reviewers, and editors in the nutrition and obesity research communities can see that the conclusion about non-monotonicity of bias as a function of covariate reliability holds only under specific and arguably contrived circumstances. Awareness of this offers some modest reassurance and also interesting insights into the design, analysis, and interpretation of studies.\n\n\nDerivation\n\nConsider the following extension to simple linear classical measurement error model\n\nAdditionally, we depict Model 1 in Figure 1 (omitting the error terms for Y, X, and Z′).\n\nSimple model including one confounder measured with error.\n\nIn Figure 1, X represents the independent variable (i.e., exposure, predictor, the putatively causal factor of interest), which could be a measured nutrient, a treatment received or not received, a personal factor, or any other measurable quantity. Z represents a confounding variable which we assume is not observed. Again, Zcould be any measurable variable, particularly an aspect of dietary intake. Random measurement error is represented bye. Here we consider classic true score phenomenon, which is a simple conceptualization of measurement error. We denote the error-contaminated measure of Z as Z′, with Z′being a simple additive function of Z and e, where e is independent of Z.\n\n“Classical test theory, also known as true score theory, assumes that each person has a true score, T, that would be obtained if there were no errors in measurement. A person’s true score is defined as the expected score over an infinite number of independent administrations of the scale. Scale users never observe a person’s true score, only an observed score …. It is assumed that observed score … = true score (T) plus some error (E) …. It is also assumed that … the expected value of such random fluctuations (i.e., mean of the distribution of errors over a hypothetical infinite number of administrations on the same subject) is taken to be 0. In addition, random errors are assumed to be uncorrelated with a true score, with no systematic relationship between a person’s true score and whether that person has positive or negative errors”.19\n\nFinally, Y represents the outcome variable (i.e., the dependent variable, the thing to be predicted).\n\n\nConfounding, direct effects in mediation, and incremental predictive validity\n\nIt is noteworthy that:\n\n(a) With minor modifications, the model depicted in Figure 1 can specify a situation of confounding, causal mediation, or correlated predictors.\n\n(b) The resulting correlation matrix among the variables depicted will be unchanged as a result of these modifications.\n\n(c) The same statistical test will be needed when testing for an association while controlling for confounding, testing for direct effects beyond mediated effects in a mediation model, or testing for incremental predictive validity.6,20,21\n\nFor example, in the model as depicted, Z is a confounder of the association between X and Y, and the test of interest is whether the squared partial correlation of X and Y after controlling for Z is not zero. However, if we reverse the direction of the arrow from Z to X (the arrow labeled with the coefficient γ), we are describing a mediation model22 in which Z is the hypothesized mediator and the test for the direct effect of X on Y can again be whether the squared partial correlation of X and Y after controlling for Z is not zero.23 As an example, Feng et al.24,p. 3336 assessed both the direct effects and “the potential indirect effect of sodium intake on blood pressure via body mass index.” That is, Feng et al. modeled BMI as the mediator of sodium intake on systolic and diastolic blood pressure and performed mediation analysis to evaluate the total effect, direct effect, and indirect effect via BMI.\n\nFinally, if we replace the arrow from Z to X with a double-headed arc,25 we are not specifying the causal relation between X and Z, only that they are correlated. In this case, we can then test whether, after accounting for the predictive ability of Z, X can add to the ability to predict Y. For example, Pichler et al.26,p. 616 aimed “to assess the accuracy and precision of a BIA [bioimpedance analysis] device and the relative contribution of BIA beyond the anthropometric parameters [emphasis added].”\n\nGiven points a, b, and c above, the implications of the methodological issue we are addressing apply to a broad swath of the research questions and hypotheses typically addressed in our field.\n\nFor simplicity, we consider that all variables are such that the use of ordinary least squares linear multiple regression (i.e., multivariable regression27) is appropriate. Extensions to other data forms are available.28 Suppose that investigators wish to test whether X is associated with (i.e., predicts) Y after controlling for Z. However, the investigators do not have measures of Z. Rather, they have measures of Z′, the error-contaminated or imperfect measurement of Z. That is why in Figure 1, Z′ is depicted in a rectangle whereas the other variables, which are assumed to be measured without error for this stylized example, are depicted in ellipses.\n\nThe question then becomes, will the type I error rate (i.e., sometimes called the false-positive rate) be maintained at its appropriate nominal levels if we control for Z′ rather than Z and how will any degree of bias in the significance testing be related to the reliability of Z′? From Figure 1, the reliability29 of Z′ is α2. If controlling for Z′ leads to unbiased inference using standard frequentist significance testing at the 0.05 significance level, then (because in this hypothetical, X has no effect on Y) the null hypothesis for the test or the association of X with Y after controlling for Z′ would yield significant results only 5% of the time. The (false) power (i.e., type-1 error rate inflation above the nominal significance level) of the test of the association of X with Y after controlling for Z′ would be the degree of bias in the significance testing procedure under these circumstances. To calculate this degree of bias or false power, we can begin by constructing the correlation matrix depicted in Table 1.\n\nThe correlation coefficients in the cells of the matrix in Table 1 are derived by simple application of the standard rules of path analysis.30 From there, we can calculate power by writing out the F-test31 of the association of X with Y after controlling for Z′. In our specific example, these yields (with N the sample size):\n\nFor any fixed sample size and specific model, the number of covariates and sample size are constant, meaning that the rightmost term of the equation becomes irrelevant if one is only concerned with the power as a function of the measurement reliability, which is our context here. Therefore, recognizing that the non-centrality parameter and the power (false power or bias) will be monotonic functions of only the left term on the right side of equation 1, [RY.X,Z′2−RY.Z′21−RY.Z′2], we can deal only with that. This term, [RY.X,Z′2−RY.Z′21−RY.Z′2], is in fact none other than the squared partial correlation coefficient (in nonmathematical terms, a squared partial correlation coefficient can be defined as the proportion of the remaining variance in the dependent variable after controlling for the covariates that can be “explained” by the independent variable of interest32) of X and Y after controlling for Z′. It is important to note that this is the squared partial correlation coefficient and not the squared semi-partial correlation coefficient [in nonmathematical terms, a squared semi-partial correlation coefficient can be defined as the proportion of the total variance in the dependent variable that can be “explained” by the independent variable of interest, after controlling for the covariates32], because the difference in the denominators of the two coefficients can be helpful in understanding the phenomena of interest in this paper. The quantities RY.X,Z′2and RY.Z′2 can then be expressed as functions of the elements of Table 1, as follows:\n\nThe squared partial correlation of Y and X controlling for (i.e., after partialing out) Z′ is:\n\nSubstituting the right sides of equations 2 and 3 into the right side of equation 4 yields:\n\nEquation 5 can then be re-expressed by substituting the zero-order correlations expressed as ‘ρ’s in terms of the path coefficients from Figure 1 as follows:\n\nOne can then calculate the squared partial correlation coefficient (the quantity in equation 6) as a function of the reliability of Z′. The reliability of Z′ is α2. The first derivative of the squared partial correlation coefficient with respect to α (the square root of the reliability coefficient) is\n\nThe first derivative of R2 with respect to α has no real roots within the open intervals α,β,γ∈(−1,1). The value of the derivative dR2dα for any value of α,β and, γ in (-1,1) is negative indicating that R2 is a decreasing function of α for fixed values of β,γ in (-1,1) (see Figure 2).\n\nAll symbolic calculations for the derivative and root solutions were performed in the computer algebra system, Wolfram Mathematica 12.0.\n\nThis shows that the squared partial correlation coefficient quantifying the association between X and Y conditional on Z′ is always decreasing in α over the half-closed interval [0,1). This means that the lower the value of α (i.e., the lower the reliability of the confounder measurement), the higher the (spurious) partial correlation of X and Y controlling for Z′ will be. This illustrates the classic concept of residual confounding. This is intuitively sensible and reassuring and refutes any implication that bias due to imperfect reliability of a confounding variable increases as the reliability increases before coming back down.\n\nIn contrast, consider the just slightly more complex model as\n\nThe model in Figure 3 is the same as that in Figure 1 except that now X is also measured with error, so we observe X′ rather than X. This then produces the 3×3 correlation matrix in Table 2.\n\nThe analogues for equations 5 and 6 from Model 1 are now equations 7 and 8 for Model 2, which are:\n\nEquation 7 can then be re-expressed substituting the zero-order correlations expressed as ‘ρ’s in terms of the path coefficients from Figure 3 as follows:\n\nFor pedagogical purposes, let us follow Westfall and Yarkoni’s approach and let: αX=αZ=α.\n\nThen, equation 8 simplifies to:\n\nOne can then calculate the squared partial correlation coefficient (the quantity in equation 9) as a function of α, or the reliability of Z′ and X′, which is α2. Taking the first derivative of the squared partial correlation coefficient with respect to the reliability coefficient, α2, we obtain:\n\nSetting the derivative equal to zero and solving for α under the constraints that α,β,γ are all in the interval (-1,1) results in a unique critical point, α∗∈(0,1). Because dR2dα is an even function of α,β,γ, there is a symmetric critical point, −α∗∈(−1,0). The region where α<0 is a reflection of the region α>0 across the y-axis; therefore, we can restrict our analysis to the region, α>0. In this region, with α>0, the derivative is positive for α<α∗ and negative for α>α∗ in the interval (0,1), verifying that the function increases before the critical point and decreases after. As a result, the squared partial correlation coefficient quantifying the association between X′ and Y conditional on Z′ is not monotonic in α over the half-closed interval [0,1). This is illustrated in Figure 4 below with (γ=0.19,β=0.50).\n\nThis would seem to support Westfall and Yarkoni’s statement that “the effect of reliability on error rates is even less intuitive: there is a non-monotonic relationship, such that type 1 error approaches 5% [the nominal rate they set] when reliability nears 0 or 1, but is highest when reliability is moderate”.6 What is not made clear in their paper is the reliability of what? Specifically, their statement is not true when referring to a single reliability coefficient. It is only true because they have constrained the reliability of the exposure measurement and the reliability of the confounder measurement to have the same value. If we decouple them (as there is no a priori reason that they must have the same value), and return to equation 8 instead of equation 9, we can see that, when Z is a confounder, the squared partial correlation coefficient (the quantity in equation 8) as a function of αZ, or the reliability of Z′, is always increasing as a function of αX, or the reliability of Z′.\n\nThis leads to the insight that it is the relative values of αZ and αX that determine this pattern. To make this clear, we re-express αX=ωαZ and then substitute into equation 8 to yield:\n\nin reduced form we have R(Y,X′.Z′)2=[(αzβωγ)2(1−αz2(2−αz2))(1−(ωαz2γ)2)(1−(αzβ)2)]\n\nprovided that ω<1/(γαz2)\n\nUsing this expression, we can make the following observations. This expression is a product of all positive terms hence is positive. If we relax the assumptions made above and only impose that αz∈(0,1) and γ and β are both set at 1.0 and ω = -1, then we can show that the partial correlation above is monotone increasing with the reliability αz and converges to ½ as αz get close to 1 from the left (αz<1). Figures 5 and 6 show the plots of the partial correlation and its first derivative, respectively, as a function of αz when γ and β are both set at 1.0 and ω = -1, both illustrating that the partial correlation is a non-decreasing function of αz in these settings. Further, it turns out that if we assume αX=ωαZ+a0 and we set ω=0.8 and a0=0.2 while keeping γ and β both at 1.0, the partial correlation is also monotone in αz(plot not shown).43 We provide a small R code to reproduce the plots presented in this paper along with the case where we set ω=0.8 and a0=0.2 while keeping γ and β both at 1.0.43\n\nWhat these derivations and illustrations show is that the degree of ‘false power’ or bias can actually goes up as one moves from a completely unreliable measure of a confounding variable to a modestly reliable measure of a confounding variable as Westfall and Yarkoni opined, but only if one artificially constrains the reliability of the exposure measurement to also be increasing as a function of the reliability of the confounder measurement. This is because, as we showed previously,33 although the numerator of a ratio and the denominator of a ratio may both move toward their “proper” values monotonically as a function of a single variable, they may do so at different rates such that the ratio may change non-monotonically.\n\n\nImplications for optimal design\n\nMany epidemiologic studies involve observational (not interventional) studies and as such are not under experimental control.34 Such observational data are often prone to measurement error, missing data, and confounding. While it is well known that measurement error associated with a single exposure, X, under some very specific circumstances, leads to attenuated associations in simple linear regression, less is known about how measurement error influences estimation, testing validity, and optimal study designs when investigators are interested in modeling the association between the exposure, and an outcome, Y, after controlling for Z, a confounder, where are all measured with error and complex error structures and analysis methods may prevail.\n\nIn nutritional studies involving estimation of dietary exposures on disease outcome models, linear regression calibration is the most applied method.35 Linear calibration-based methods to measurement error correction involve two-stage models where replacement values for the true dietary exposure are simulated or obtained from its conditional distribution given the measured value and association between the simulated measures of the exposures and the outcome are estimated in the second stage.36 Under the linear calibration approaches, non-differential measurement error and uncorrelated errors between the dietary and reference instruments are assumed.35 In models where a confounder and an exposure are both measured with error or when multiple covariates are measured with error, multivariable regression calibration is often used in nutritional studies.37 The multivariable regression calibration approach is performed under assumptions of random within-person errors or under combinations of random error. In general, the effect of measurement error in a single mis-measured or imprecisely observed exposure is to attenuate its effects on health outcomes. However, how measurement error in both a confounder and exposure influence their estimated effects on the dependent variable is less clear. Measurement error can modify or mask the effects of a confounder when both the confounder and exposure are error prone.34\n\nWhile efforts have been made to develop statistical approaches that correct for measurement error to reduce biases in the estimation of exposure effects, less has been done to determine how the presence of measurement error in an exposure or in both exposure and confounder influences optimal study designs. By optimal study design, we mean the design which minimizes some loss function (e.g., some weighted function of quantities of interest such as power, type 1 error rate, bias, financial cost, study duration, etc.) subject to user-defined constraints.38 Spiegelman and Gray39 developed cost-efficient and statistically powerful cohort study designs for continuous exposures measured with error in binary logistic regression models. The authors proposed three ways to optimize study designs in the presence of errors. These include the inclusion of an internal validation study, external validation of subsamples obtained from other studies, and the use of better exposure methods for assessment.39\n\nSuch thinking invites several questions for future research when we wish to test for the association between an independent variable and a dependent variable after controlling for a third variable, and all may be measured with error.\n\n• A first question is a meta-research question40: What is the state of practice? When investigators apply measurement error correction procedures, do they typically do so for X, Z, Y, or some combination? Although systematic reviews of related questions have appeared,35 we are not aware of one that has answered this exact question.\n\n• A second question is how does applying such measurement error corrections affect power and bias if applied to various combinations of X vs Y vs Z?\n\n• Third, do the answers to the second question suggest under which circumstances, to what extent, and for which variables (X, Y, or Z) investigators should invest their resources in improving the reliability of if the possibility of increasing reliability of each at the cost of finite funds exists? Such inquires might lead to the counterintuitive finding that under some circumstances one should invest more in studying the nuisance variables one is not interested in than the putative causal factors in which one is interested.\n\n\nGeneral implications\n\nWithout knowing the exact nature of any phenomenon or set of phenomena we are studying, we cannot know exactly which situation will prevail. If we did, we would not need to undertake the research. However, we can say from general knowledge of the measurement properties of variables frequently used in nutrition and epidemiologic research, and of the magnitude of associations typically studied in epidemiology and nutrition and obesity, that the hypothetical scenarios we have evaluated above are within the realm of the typical association study. Therefore, it seems plausible that not only is measurement error a substantial concern in our models but so are statements that may be patently untrue, such as that an association actually becomes more significant when controlling for a plausible confounder or that measurement error only attenuates effects. We thus may need to take observational research with more than a considerable grain of salt when drawing causal inferences. Yet further, the plausibility of our hypothetical example suggests the importance of not merely acknowledging measurement error as most of us do in our articles on nutrition epidemiology, but actually building formal measurement error corrections into the analysis as described elsewhere.41 Included in overall implications for research in nutritional epidemiology are the untrue yet-seeming implicit default assumptions that: 1) there is a linear or monotonic dose-response of risk with intake of a food or nutrient; 2) confounders act independently of one another, additively, linearly, with predictable effect, when they clearly do not, accentuating concerns about the ignoring of potential interactions of multiple confounders that are either not measured at all or not measured accurately42; and 3) unmeasured or poorly measured confounders can be dismissed as serious threats to statistical conclusion validity because it is implausible that a confounder or set of confounders could imbue sufficient bias to produce the observed association, when in fact large samples produce large power to mistakenly detect small biases as actual associations or effects. Finally, our illustrations and derivations point out the importance of evaluating the quality of our procedures and improving them when possible.\n\nAs outlined in the beginning, the topic of measurement error in covariates has critical application for nutritional epidemiology around the practice of determining nutrient and energy intake and using it for developing nutritional guidance and policies. It is unclear what role interpretations based on inaccurate measurements have played in national and local health-related policies or health care guidance for individuals. It should be recognized that a great need exists for accurately determining nutrient or energy intake. This need will not become a priority if we continue to accept inaccurate data as “good enough” for the purpose. Therefore, moving forward, we need to discontinue the use of methods that generate decidedly inaccurate data. Next, we urge that concerted efforts be made in redirecting resources to developing methods that objectively measure nutrient and energy intake with high fidelity and for long periods of time. Until such time, we need to be prepared to accept the counterintuitive conclusion that under some circumstances, in our efforts to improve the reliability or power of our methods, sometimes nothing is better than something.\n\n\nEthics and consent\n\nEthical approval and consent were not required.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: rszoh/ReliabME: Version 1. https://zenodo.org/doi/10.5281/zenodo.12639728. 43\n\nAnalysis code available from: https://github.com/rszoh/ReliabME/.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors acknowledge the reviewers for their comments which help improve the presentation of the paper. The authors also acknowledge Jennifer Holmes, ELS, for language editing and editorial assistance.\n\n\nReferences\n\nMcHenry EW, Ferguson HP, Gurland J: Sources of error in dietary surveys. Can. J. Public Health. 1945; 36(9): 355–361. Reference Source\n\nKlurfeld DM, Hekler EB, Nebeker C, et al.: Technology innovations in dietary intake and physical activity assessment: challenges and recommendations for future directions. Am. J. Prev. Med. 2018; 55(4): e117–e122. Publisher Full Text\n\nWoteki CE: Measuring dietary patterns in surveys. Vital Health Stat. 1992; 4(27): 101–108.\n\nLear S: Should you eat red meat? Navigating a world of contradicting studies.2019 Oct 11 [Cited 2021 Dec 13.]. Reference Source\n\nDhurandhar NV, Schoeller D, Brown AW, et al.: Energy Balance Measurement Working Group. Energy balance measurement: when something is not better than nothing. Int. J. Obes. 2015; 39(7): 1109–1113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWestfall J, Yarkoni T: Statistically controlling for confounding constructs is harder than you think. PLoS One. 2016; 11(3): e0152719. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGadbury GL, Allison DB: Inappropriate fiddling with statistical analyses to obtain a desirable p-value: tests to detect its presence in published literature. PLoS One. 2012; 7(10): e46363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaplan RM, Chambers DA, Glasgow RE: Big data and large sample size: a cautionary note on the potential for bias. Clin. Transl. Sci. 2014; 7(4): 342–346. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown AW, Aslibekyan S, Bier D, et al.: Toward more rigorous and informative nutritional epidemiology: the rational space between dismissal and defense of the status quo. Crit. Rev. Food Sci. Nutr. 2021; 63: 3150–3167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeadows A, Higgs S: A bifactor analysis of the Weight Bias Internalization Scale: what are we really measuring? Body Image. 2020; 33: 137–151. PubMed Abstract \| Publisher Full Text\n\nHobbs M, Green M, Roberts K, et al.: Reconsidering the relationship between fast-food outlets, area-level deprivation, diet quality and body mass index: an exploratory structural equation modelling approach. J. Epidemiol. Community Health. 2019; 73(9): 861–866. PubMed Abstract \| Publisher Full Text\n\nVainik U, García-García I, Dagher A: Uncontrolled eating: a unifying heritable trait linked with obesity, overeating, personality and the brain. Eur. J. Neurosci. 2019; 50(3): 2430–2445. PubMed Abstract \| Publisher Full Text\n\nHeino MTJ, Knittle K, Haukkala A, et al.: Simple and rationale-providing SMS reminders to promote accelerometer use: a within-trial randomised trial comparing persuasive messages. BMC Public Health. 2018; 18(1): 1352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVainik U, Meule A: Jangle fallacy epidemic in obesity research: a comment on Ruddock et al. (2017). Int. J. Obes. 2018; 42(3): 585–586. PubMed Abstract \| Publisher Full Text\n\nRuddock HK, Christiansen P, Halford JCG, et al.: Response to ‘Jangle fallacy epidemic in obesity research: A comment on Ruddock et al. (2017).’. Int. J. Obes. 2018; 42(3): 586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThibodeau PH, Uri R, Thompson B, et al.: Narratives for obesity: effects of weight loss and attribution on empathy and policy support. Health Educ. Behav. 2017; 44(4): 638–647. PubMed Abstract \| Publisher Full Text\n\nJacka FN: Nutritional psychiatry: where to next? EBioMedicine. 2017; 17: 24–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrentice RL: Dietary assessment and opportunities to enhance nutritional epidemiology evidence. Ann. Intern. Med. 2020; 172(5): 354–355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCappelleri JC, Jason Lundy J, Hays RD: Overview of classical test theory and item response theory for the quantitative assessment of items in developing patient-reported outcomes measures. Clin. Ther. 2014; 36(5): 648–662. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacKinnon DP, Krull JL, Lockwood CM: Equivalence of the mediation, confounding and suppression effect. Prev. Sci. 2000; 1(4): 173–181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVickers AJ, Cronin AM, Begg CB: One statistical test is sufficient for assessing new predictive markers. BMC Med. Res. Methodol. 2011; 11: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFairchild AJ, McDaniel HL: Best (but oft-forgotten) practices: mediation analysis. Am. J. Clin. Nutr. 2017; 105(6): 1259–1271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFairchild AJ, MacKinnon DP, Taborga MP, et al.: R2 effect-size measures for mediation analysis. Behav. Res. Methods. 2009; 41: 486–498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng Q, Yang Z, May M, et al.: The role of body mass index in the association between dietary sodium intake and blood pressure: a mediation analysis with NHANES. Nutr. Metab. Cardiovasc. Dis. 2021; 31(12): 3335–3344. PubMed Abstract \| Publisher Full Text\n\nOlobatuyi ME: A user’s guide to path analysis. Lanham, Md: University Press of America; 2006.\n\nPichler GP, Amouzadeh-Ghadikolai O, Leis A, et al.: A critical analysis of whole body bioimpedance spectroscopy (BIS) for the estimation of body compartments in health and disease. Med. Eng. Phys. 2013; 35(5): 616–625. PubMed Abstract \| Publisher Full Text\n\nHidalgo B, Goodman M: Multivariate or multivariable regression?. Am. J. Public Health. 2013; 103(1): 39–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCullagh P, Nelder JA: Generalized linear models. 2nd ed.Routledge; 1983. Publisher Full Text\n\nPiedmont RL: Reliability coefficient.Michalos AC, editor. Encyclopedia of quality of life and well-being research. Dordrecht, Netherlands: Springer; 2014. Publisher Full Text\n\nAlwin DF, Hauser RM: The decomposition of effects in path analysis. Am. Sociol. Rev. 1975; 40(1): 37–47. Publisher Full Text\n\nSmithson M: Correct confidence intervals for various regression effect sizes and parameters: the importance of noncentral distributions in computing intervals. Educ. Psychol. Meas. 2001; 61(4): 605–632. Publisher Full Text\n\nAbdi H: Part (semi partial) and partial regression coefficients.Salkind N, editor. Encyclopedia of measurement and statistics. Sage; 2007; pp. 1–9.\n\nAllison DB, Heo M, Flanders DW, et al.: Examination of “early mortality exclusion” as an approach to control for confounding by occult disease in epidemiologic studies of mortality risk factors. Am. J. Epidemiol. 1997; 146(8): 672–680. PubMed Abstract \| Publisher Full Text\n\nThomas D, Stram D, Dwyer J: Exposure measurement error: influence on exposure-disease relationships and methods of correction. Annu. Rev. Public Health. 1993; 14(1): 69–93. PubMed Abstract \| Publisher Full Text\n\nBennett DA, Landry D, Little J, et al.: Systematic review of statistical approaches to quantify, or correct for, measurement error in a continuous exposure in nutritional epidemiology. BMC Med. Res. Methodol. 2017; 17: 146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFuller WA: Measurement error models. John Wiley & Sons; 2009; vol. 305. .\n\nRosner B, Spiegelman D, Willett WC: Correction of logistic regression relative risk estimates and confidence intervals for random within-person measurement error. Am. J. Epidemiol. 1992; 136(11): 1400–1413. PubMed Abstract \| Publisher Full Text\n\nAllison DB, Allison RL, Faith MS, et al.: Power and money: designing statistically powerful studies while minimizing financial costs. Psychol. Methods. 1997; 2(1): 20–33. Publisher Full Text\n\nSpiegelman D, Gray R: Cost-efficient study designs for binary response data with Gaussian covariate measurement error. Biometrics. 1991 Sep; 47(3): 851–69. [Erratum in: Biometrics 1991 Dec;47(4):1641.]. PubMed Abstract \| Publisher Full Text\n\nIoannidis JPA: Meta-research: why research on research matters. PLoS Biol. 2018; 16(3): e2005468. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurillo AL, Affuso O, Peterson CM, et al.: Illustration of measurement error models for reducing bias in nutrition and obesity research using 2-d body composition data. Obesity (Silver Spring). 2019; 27(3): 489–495. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWyss R, Lunt M, Brookhart MA, et al.: Reducing bias amplification in the presence of unmeasured confounding through out-of-sample estimation strategies for the disease risk score. J. Causal Inference. 2014; 2(2): 131–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nrszoh: rszoh/ReliabME: Version 1 (v1.0.0). [Dataset]. Zenodo. 2024. Publisher Full Text" }	[ { "id": "336327", "date": "11 Nov 2024", "name": "Nicola Bondonno", "expertise": [ "Reviewer Expertise Nutritional epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe final sentence of the introduction suggests that the target audience for this paper includes researchers, reviewers, and editors in nutrition and obesity research. However, the level of statistical detail makes the content accessible only to those with an advanced statistical background. As a nutritional epidemiologist with a strong grasp of statistics, I struggled to follow the derivations from the first sentence onward. Given this, I will not comment on the technical aspects of the math. However, as someone within your target audience, I found that the paper lacked clear takeaways, which could be improved for better accessibility and impact. I'll provide a few suggestions that may help enhance clarity.\nThe figures are informative, yet the material could be made even more approachable with a consistent, specific example that guides both experts and non-experts throughout the paper. For instance, the approach taken in Tomova et al. (2022) may serve as a useful model, where a single example runs consistently across sections (refer 1). Currently, you use a few different examples, but consolidating to one example could help maintain continuity and comprehension.\nIf I understand correctly, the association shown in Figures 4, 5, and 6 (as proposed by Westfall and Yarkoni) only holds when the reliability of the exposure increases as the reliability of the confounder increases. This isn’t typically expected in real-world settings and clarifying this point with an example would enhance understanding. In the introduction, you state, “Here we provide an explanation of the phenomenon in simple scenarios so that researchers, reviewers, and editors in the nutrition and obesity research communities can see that the conclusion about non-monotonicity of bias as a function of covariate reliability holds only under specific and arguably contrived circumstances.” While this is a valuable aim, I didn’t find this point sufficiently addressed in the Discussion. Your conclusions focus primarily on the need to improve the accuracy of exposure and confounder measurements, which is important but misses practical advice for researchers working with pre-collected data, where measurement error is often unknown. Can you clarify for readers in this situation—what should they do or acknowledge in their studies? Expanding on this in the Discussion would make your conclusions more actionable.\nI also disagree with the statement: “Included in overall implications for research in nutritional epidemiology are the untrue yet-seeming implicit default assumptions that: 1) there is a linear or monotonic dose-response of risk with intake of a food or nutrient.” Most quality papers in the field do not assume this and instead use splines or categorize exposures to avoid such assumptions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "330829", "date": "26 Nov 2024", "name": "Tolulope Sajobi", "expertise": [ "Reviewer Expertise Biostatistics", "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\nThank you for the opportunity to review the manuscript by Zoh and colleagues on the implications of adjusting for covariates measured with error on strength of association between an exposure and covariates. The authors, through simple but elegant mathematical derivations demonstrated the implications of measurement error on Type I error and bias in the association between an exposure (e..g, nutrient) and outcome. Specifically, the authors examined the impact of power and bias under two scenarios\nWhen the confounding variable Z is measured with error but not the exposure variable X. When the confounding variable Z and exposure variable X are both measured with errors.\nThe authors showed that there is a non-monotonic relationship between the degree of measurement error and the Type I error and bias in the estimation of the association between an exposure and outcome. The authors also outlined some ideas for future research.\nAs a minor revision, the authors should comment on the possible conclusion from extending this investigation to generalized linear models, especially with a non-linear link function.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/13-827
https://f1000research.com/articles/11-1388/v1	25 Nov 22	{ "type": "Research Article", "title": "Characteristics and contributing factors of adverse drug reactions: an analytical study of patients with tuberculosis receiving treatment under the National TB Program of India", "authors": [ "Harsh Shah", "Sandul Yasobant", "Jay Patel", "Priya Bhavsar", "Somen Saha", "Yogesh Patel", "Deepak Saxena", "Anish Sinha", "Sandul Yasobant", "Jay Patel", "Priya Bhavsar", "Somen Saha", "Yogesh Patel", "Deepak Saxena", "Anish Sinha" ], "abstract": "Background Tuberculosis (TB) continues to pose a serious threat to the public health system in India. Although the National Tuberculosis Elimination Program (NTEP) is providing a wide range of interventions from early diagnosis to complete treatment to reduce morbidity and mortality from TB, adverse drug reactions (ADR) remain a challenge in treatment adherence and completion. Methods An observational cross-sectional study was conducted in selected districts of Gujarat state. A total of 593 reported TB patients were recruited with an adjusted unified distribution based on the type of cases, site of diseases, and service facility through a simple random sampling method. A semi-structured questionnaire tool was used to collect socio-demographic, clinical, and ADR-related data from the TB patients. Data was analyzed for the frequency, percentage, chi-squared, and adjusted odds ratio to find the association between the variables. Results\nThe majority of the study participants were male (87.2%), aged 15 to 60 (57.8%), daily laborers (22.4%), and married (64.2%). Over 75% of individuals had pulmonary TB, with 87% having experienced their first episode, 83% being new cases, and 44.7% having a history of addiction. ADR with mild symptoms was reported by more than a quarter (29%) of TB patients during the intensive phase (77%). The association between ADR experience and drug susceptibility was significant (p<0.005) and drug-resistant TB patients experience two times more ADRs than drug-sensitive TB patients (OR 2.04). Binomial logistic regression was carried out to describe the association between various variables and occurrence of ADRs. Conclusion\nThe study highlighted a need to enhance health care providers’ capacity and program structure for managing ADRs among TB patients. In order to completely eliminate TB across the country, it also emphasized the attention for a holistic and all-encompassing strategy for managing TB patients at the field level.", "keywords": [ "Tuberculosis", "Adverse Drug Reactions", "National TB Program", "India" ], "content": "1. Introduction\n\nTuberculosis (TB) is a communicable disease that remains a major cause of illness and death across low and middle-income countries (LMIC) even after the discovery of novel diagnostic methods and chemotherapeutic drugs. The incidence of TB and rising numbers of multidrug-resistant TB cases are still a concern for countries with high disease burden. As per the global TB report, the incidence of TB in India is approximately 2.8 million cases annually, accounting for almost a quarter of all TB cases worldwide. Even though a six-month drug regimen can successfully treat about 85% of those who develop TB, TB remains a significant threat to public health systems due to difficulties in early detection and the required treatment duration.1 Over the years, the National TB Elimination Program (NTEP) has expanded the range of anti-tuberculosis therapy (ATT) drugs utilized in daily regimens and revised programmatic guidelines for the management of drug-resistant TB.2\n\nThe critical component of ATT is the standard directly observed treatment, short course (DOTS) chemotherapy regimen for drug-susceptible TB and the extended multidrug regimen for drug-resistant TB, depending on the culture and drug susceptibility tests. Poor treatment adherence increases the risk of drug resistance, treatment failures, relapses, and deaths. The persistence of infection among TB patients due to poor adherence continues to be a barrier to the success of TB programs.3 To avoid morbidity, mortality, and the spread of TB, every effort should be made to persuade and motivate patients to continue their treatments despite any discomforts due to adverse drug events (ADEs). Almost all anti-TB medications result in adverse drug reactions (ADRs) that can range in severity from minor to fatal. Compared to second-line treatments, first-line anti-TB medications are often well tolerated by patients. These ADRs can cause TB patients to stop their therapy, resulting in needless morbidity, drug resistance, treatment failure, a decreased quality of life, or even death.4–6 Comorbid conditions and risk factors influence the incidence of ADR and the outcome of TB treatment.\n\nBetween 8 and 85% of patients experience different side effects, ranging from mild to severe.5 About 10–25% of patients who experience side effects develop significant and deadly medication reactions or serious adverse events (SAEs).7–9 Treatment failure, relapse, or the formation of resistance are risks for patients who take their drugs inconsistently or stop taking them due to side effects.10–13 It is crucial that all TB patients receiving therapy effectively manage and keep track of ADRs, especially major ones. Early ADR detection and prompt care can improve drug compliance, improve the treatment outcome, and stop the emergence of drug resistance.14 Due to their under-recording and under-notification when monitored by the NTEP, the range and characteristics of ADR are not well recognized. With this background, the present study was conducted to assess the prevalence and characteristics of ADRs among TB patients and identify various epidemiological, socio-demographic, and programmatic factors associated with ADRs in the Western state of India, Gujarat.\n\n\n2. Methods\n\nA descriptive observational cross-sectional study was conducted from 3rd May 2021 to 30th July 2021 in the Western state of India, Gujarat. The study was conducted through the district tB centre (DTC) and 32 tuberculosis units (TUs) in Gandhinagar and Surat districts (Gujarat state), with TB patients registered and managed. NTEP has been implemented in all districts of the state. Each district has a district TB center, which monitors the program for the entire district. The district is further divided into sub-districts i.e., TUs, at each block. Under the TUs, outlying peripheral (government and private) health facilities (PHI) provide programmatic management for TB patients.\n\nThe assessment targeted a diverse profile of TB patients, such as drug-sensitive TB (DSTB), drug resistance TB (DRTB), pediatric TB, and extra-pulmonary TB. It included both public and private sector patients. The patients diagnosed with TB are reported on the online digital patient management portal Nikshay in the notification registers by the health facilities.15 The list of reported TB patients from 1st July 2018 to 31st December 2020 was extracted from Nikshay to ensure that the study population completed treatment based on the duration of the treatment regimen. A total of 20,668 patients were reported in the Nikshay portal from both districts during that period.\n\nThe sample size was calculated based on the formula of N=Z21−α/2P(1−P)/ε2, where N=sample size, Z21−α=confidence interval, P=estimated proportion, ε=desired precision/error, with estimated proportion of 50% of ADR occurrences. Based on sample size calculation, it was derived that over 534 TB patients had to be included in the study to have a confidence level of 98% and a desired error that is within ±5% of the measured/surveyed value. Additionally, the final sample size accounted for around a ~10% non-response rate, bringing the number of study participants to about 593. The eligible TB cases were listed with the inclusion and exclusion criteria below. Inclusion criteria: the TB patients reported through Nikshay, their current state PHI was within the selected geographical areas of Gujarat state, and they were given treatment. Exclusion criteria: TB patients who migrated or were untraceable or did not reside in the current PHI surveyed areas or whose relatives didn't provide consent were excluded from the study.\n\nFrom each TU, patients were recruited randomly depending on their availability and willingness to participate. Simple random sampling was adopted to select TB cases within the selected geographic areas until the saturation of the sample size. However, a proportionate adjustment based on the type of cases, service facility, and site of disease was considered for the unified distribution across the study geography to ensure the collective representation of the study participants.\n\nA semi-structured interview followed by a semi-structured, pilot-tested ADR assessment questionnaire was used to collect the data in the vernacular language (Gujarati). A pretested and semi-structured questionnaire tool consisting of information regarding primary socio-demographics, medical history, history of addiction and comorbidity, and information about the grade and type of ADRs was administered by the trained researchers in the vernacular language through personal interviews by undertaking home visits. The researchers were trained to administer the questionnaire with a participatory approach and role play to prepare them to interview the study participants for the required information.\n\nThe World Health Organization (WHO) has defined adverse drug reactions (ADRs) as “A response to a drug which is noxious and unintended, and which occurs at doses normally used in humans for the prophylaxis, diagnosis, or therapy of disease, or the modification of physiological function”.16 The cornerstones of DSTB therapy continue to be a treatment plan with a minimum duration of six months and numerous first-line medicines (FLDs), such as isoniazid (H), rifampicin (R), pyrazinamide (Z), ethambutol (E), and streptomycin (S). Similar to this, NTEP offers streamlined regimens for several forms of DR-TB, including shorter oral bedaquiline-containing MDR/rifampicin resistant-TB regimens and longer oral M/XDR-TB (mono or extreme drug resistant) regimens generally ranging from six to nine months but can reach up to 20 months. The drug dosages are adjusted based on the age, weight, severity of the disease, site of the disease, and type of drug resistance/susceptibility towards ATTs.\n\nOnce the data collection was completed, data sets were scrutinized for completeness and validation by the different sets of the researchers. The study participants were contacted again if any data variables were found to be missing by the researchers who had collected the primary data. The patient data on various variables was tabulated, analyzed, and interpreted by proper statistical methods using IBM SPSS statistics software version 20 (RRID:SCR_019096). The chi-squared test was used to compare groups, while the chi-squared for the trend examined linear trends. Risk measures were determined using odds ratios (OR) and 95% confidence intervals (CI). Crude OR and 95% CI were calculated for the interpretation of univariate analysis, with the level of significance set at p<0.05. To identify the independent factors associated with ADRs, adjusted odds ratios (AOR) and 95% CI were calculated by bivariate logistic regression analysis.\n\n\n3. Results\n\nBased on the study criteria, 105 (18%) TB patients from Gandhinagar and 488 (82%) from Surat were included. There were 536 (90%) patients who completed the treatment and 57 (10%) on treatment.\n\nThe mean age of study participants was 34.6±15.6 years, and the median age was 31 years, ranging from 1 to 85 years. The majority of study cases, 517 (87.2%), were in the age group of 15–60. There were 343 (57.8%) male patients and 250 (42.2 %) female patients. There were 99 (16.7%) illiterates, 52 (8.8%) graduates, and 133 (22.4%) daily laborers and 381 (64.2%) of the patients were married. The association between age categories, marital status, and education status and adverse drug reaction was not significant (p>0.05). However, among gender and occupation status, it was found to be significant (p<0.05) (Table 1).\n\nThe study participants were comprised of 147 (25%) extra-pulmonary TB (EPTB) patients and 446 (75%) pulmonary TB patients (PTB), 519 (87%) of whom had contracted the first episode of TB. The distribution of the type of cases as per national guidelines was 492 (83%) new cases and 69 (12%) previously treated cases on the drug-sensitive TB treatment regimen, while 32 (5%) cases were on the drug-resistant treatment regimen. A total of 66 (11%) TB patients were receiving treatment from private providers. The study reported that 268 (44.7%) had a history of addiction, with 91% addicted to tobacco (either smokeless or smoking) and 9% to alcohol. Among them, 41% had an addiction to tobacco and alcohol, while 1.2% had addictions to psychotic substances. Sixty-one (10%) reported the presence of at least one comorbidity, while the major contribution of comorbidity was diabetes (50%). The number of patients with a HIV co-infection was deficient in numbers to be included in a detailed analysis.\n\nDuring the study, it was observed that 172 (29%) patients experienced ADRs with at least one symptom. Out of those, 80% had mild symptoms, and 133 (77%) experienced them during the early (intensive) phase of the treatment initiation. The 18 (56%) drug-resistant TB patients on second-line ATTs reported ADR, 50% of whom reported moderate and severe ADRs. The association between ADR experience and drug susceptibility was significant (p value of 0.005; Chi-squared 12.193) and drug-resistant TB patients experience two times more ADRs than drug-sensitive TB patients (OR 2.049, CI: 1.47–2.86). The TB patients had experienced gastric disturbances, skin-related symptoms, peripheral nervous system symptoms, arthralgia, ophthalmic discomfort, and psychological disorders during ATT (Table 2).\n\nThe study used a binomial logistic regression model to estimate the bivariate odds ratio and a 95% confidence interval to describe the association between predictor variables and the occurrence of ADRs. The study used the dataset's socio-demographic, clinical, and programmatic service delivery variables to develop the predictive model. The model showed that gender, drug susceptibility status, and history of addiction were statistically significant (p<0.05). The regression model showed that the Nagelkerke R2 value was 0.139 with a classification accuracy of 71% (Table 3).\n\n\n4. Discussion\n\nAdverse events, defined as any unfavorable medical occurrence, can also be linked to treatment with these medications but are not always causally related. The study was conducted in only a selected part of the country, but the findings of the study provide insight into the drug reactions observed by TB patients during the course of treatment. The present study revealed that the prevalence of ADRs was 29% among the study population, similar to various worldwide studies ranging from 8% to 85%.3–6,14,17–20 Several studies reported more ADR prevalence in drug-resistant TB patients, similar to the present study, where 50% of DRTB patients experienced ADRs.19,21–23 The variance in ADR prevalence between these studies could be due to several data collection variables including the ADR reporting mechanism, patient-reported (subjective) or clinician-detected (objective), and variations in the use of particular anti-tubercular drugs such as dosage and ancillary medications used for ADR management.\n\nThe study observed 71% mild grade ADRs, 77% of which occurred in the early period (intensive phase) of treatment. Several studies also reported that major or severe ADRs were less common (occurring in approximately 2% of the cases, reaching 8% in specialized clinics), and ADRs were more prevalent in the intensive phase than in the continuation phase.24–27 Many studies have reported the frequency of symptoms and types of ADRs, which can range in severity from mild to severe, caused by both first-line and second-line anti-TB medications. The drug-specific ADRs may cause either a reduction of dosage or termination of the offending drug(s) and lead to common ADRs up to organ-specific toxicity in severe cases.3,21 The present study reported that the most common ADRs were gastric discomfort and arthralgia, followed by cutaneous ADRs, peripheral neuropathy, ophthalmic photosensitivity, and psychiatric disorders (headache/anxiety/confusion), similar to various studies conducted in India.22,28–30 When compared to other adverse events, patients report gastrointestinal adverse events and arthralgia more frequently, which can contribute to subjective variation and a high prevalence of these events.\n\nA study from Uttar Pradesh by Prasad et al. and Gujarat by Jakasania et al. reported that there was no statistically significant difference in patients suffering from ADRs concerning variables such as age group, gender, educational and occupational status, history of addiction, and presence of a comorbidity, episodes of TB, and healthcare facility opted for services.19,29 According to the study, one of the associated factors for ADR is the female gender. However, we may not have observed this since most participants in our study cohort were men. The logistic regression model of the present study identified that gender, drug susceptibility, and history of addiction were each predisposing risk factors for ADRs.\n\nThe present study recorded ADRs or adverse events from the history of patients that could lead to subjective variations and recall bias. The type and grade of ADRs were also recorded from the patient's perspective, limiting the researchers to identify the drug-specific symptom of ADRs. In the absence of patients' medical records, the study could not assess or record the nutritional status at the time of ATT, the severity of comorbidity, or confirm hospitalisation due to severe ADRs. This was one of the reasons that during the study, researchers had not considered the history of stoppage of the offending drug(s), alterations in the treatment regimen, or management received for the discomfort. The study excluded one TB patient who was non-traceable at the time of the data collection. The excluded TB patient could have provided additional information that support the results.\n\n\n5. Conclusions\n\nThe present study focused on adverse events pertaining to TB patients missed by the health system. The analysis delivered crucial conclusions that could direct policymakers to educate and train all healthcare professionals and high-risk patients on how to solicit and manage ADRs among patients receiving programmatic treatment effectively. It is crucial to strengthen the program by carefully examining treatment plans based on medical history, ensuring treatment compliance, managing adverse events aggressively and proactively, and establishing a training cascade for health care providers and treatment supporters.\n\n\nEthics and informed consent\n\nEthical approval for this study was obtained from the Indian Institute of Public Health Gandhinagar- Institutional Ethics Committee (TRC-IEC No:18/2020-21). The administrative approval to conduct the study was received from the State TB cell, Department of Health and Family Welfare, Government of Gujarat.\n\nWritten informed consent for publication of the patients’ details was obtained from the patient or the parents/guardian of the participant if they were under 18 years of age.\n\n\nAuthor contributions\n\nAll authors contributed equally to the development of this study. All authors contributed to data analysis, drafting, or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.", "appendix": "Data availability\n\nFigshare: A Study on Adverse Drug Reaction Among TB Patients, https://doi.org/10.6084/m9.figshare.21185875.v1. 31\n\nThe project contains the following underlying data:\n\n- Gujarat Baseline Data for Online Publication.xlsx\n\n- Protocol Methodology for the study assessing the Adverse Drug Reactions among TB patients.docx\n\n- ADR Questionnaire Tool - Gujarati.docx (in the language in which the questionnaire was distributed)\n\n- ADR Questionnaire Tool.docx (in English)\n\nFigshare: STROBE checklist for ‘Characteristics and contributing factors of adverse drug reactions: an analytical study of patients with tuberculosis receiving treatment under the National TB Program of India’, https://doi.org/10.6084/m9.figshare.21185875.v1. 31\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe wish to extend our gratitude to the Health and Family Welfare, the Government of Gujarat & Jharkhand, and State TB Training and Demonstration Center (STDC) team and the National Tuberculosis Elimination Program (NTEP) staff for their kind support during the project activities. We are thankful to the Dr Satish Makwana (State TB Officer), Dr Dipak Patel and Dr Sheladia (District TB Officers), all the NTEP staff and all the participants of Gujarat for providing the necessary information for this study. We are also thankful to the research team of the Indian Institute of Public Health Gandhinagar (IIPHG) for their support during the field activities. We thank the World Health Partners (WHP) for their continuous support in the Closing the Gaps in TB Care Cascade (CGC) project. We express our sincere thanks to USAID New Delhi, India, for funding this study as a part of the larger project, namely, Closing the gaps in the TB care cascade.\n\n\nReferences\n\nSingh S, Dey B, Sachdeva K, et al.: Challenges in Tuberculosis Diagnosis and Management: Recommendations of the Expert Panel. J. Lab. Physicians. 2015; 7(01): 001–003. Publisher Full Text\n\nKhaparde S: The national strategic plan for tuberculosis step toward ending tuberculosis by 2025. J. Mahatama Gandhi Inst. Med. Sci. 2019; 24(1): 17. Publisher Full Text\n\nSingla R, Sarin R, Khalid UK, et al.: Seven-year DOTS-Plus pilot experience in India: results, constraints and issues. Int. J. Tuberc. Lung Dis. 2009; 13: 976–981. PubMed Abstract\n\nJayapriya B, Antony LJ, Balamurugan PV, et al.: Pattern of adverse drug reactions of antitubercular drugs in tuberculosis patients with comorbidities and risk factors in South Indian government health-care facilities. Natl. J. Physiol. Pharm. Pharmacol. 2021.\n\nSingh A, Prasad R, Balasubramanian V, et al.: Prevalence of adverse drug reaction with first-line drugs among patients treated for pulmonary tuberculosis. Clin. Epidemiol. Glob. Health. 2015; 3: S80–S90. Publisher Full Text\n\nBorisov S, Danila E, Maryandyshev A, et al.: Surveillance of adverse events in the treatment of drug-resistant tuberculosis: First global report. Eur. Respir. J. 2019 Dec 1; 54(6): 1901522. PubMed Abstract \| Publisher Full Text\n\nSaha A, Margaret Shanthi FX, Blessed Winston A, et al.: Prevalence of hepatotoxicity from antituberculosis therapy: A five-year experience from South India. J. Prim. Care Community Health. 2016; 7(3): 171–174. PubMed Abstract \| Publisher Full Text\n\nTost JR, Vidal R, Caylà J, et al.: Severe hepatotoxicity due to anti-tuberculosis drugs in Spain. Int. J. Tuberc. Lung Dis. 2005; 9(5): 534–540. PubMed Abstract\n\nYee D, Valiquette C, Pelletier M, et al.: Incidence of Serious Side Effects from First-Line Antituberculosis Drugs among Patients Treated for Active Tuberculosis. Am. J. Respir. Crit. Care Med. 2003 Jun 1; 167(11): 1472–1477. PubMed Abstract \| Publisher Full Text\n\nAlipanah N, Jarlsberg L, Miller C, et al.: Adherence interventions and outcomes of tuberculosis treatment: A systematic review and meta-analysis of trials and observational studies. PLoS Med. 2018; 15(7): e1002595. PubMed Abstract \| Publisher Full Text\n\nNahid P, Dorman SE, Alipanah N, et al.: Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis. Clin. Infect. Dis. 2016; 63: 853–867. PubMed Abstract \| Publisher Full Text\n\nPettit AC, Cummins J, Kaltenbach LA, et al.: Non-adherence and drug-related interruptions are risk factors for delays in completion of treatment for tuberculosis. Int. J. Tuberc. Lung Dis. 2013; 17(4): 486–492. PubMed Abstract \| Publisher Full Text\n\nAwofeso N: Anti-tuberculosis medication side-effects constitute major factor for poor adherence to tuberculosis treatment. Bull. World Health Organ. 2008; 86: 240–24D. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrasad R, Singh A, Gupta N: Adverse drug reactions in tuberculosis and management. Indian J. Tuberc. 2019; 66: 520–532. Publisher Full Text\n\nKumar S: STDHIDNGovt of I. NIKSHAY- A Web-based Solution for Monitoring of TB Patients.[cited 2022 May 27].Reference Source\n\nWHO: Safety of Medicines: A guide to detecting and reporting adverse drug reactions. Geneva:World Health Organization;2002. 2002.2(2002).\n\nSant´Anna FM, Araújo-Pereira M, Schmaltz CAS, et al.: Adverse Drug Reactions Related to Treatment of Drug-Susceptible Tuberculosis in Brazil: A Prospective Cohort Study. Frontiers in Tropical Diseases. 2022; 2: 2. Publisher Full Text\n\nPhillipson J, Kuruppu N, Chikura T, et al.: Adverse effects and duration of treatment of TB in Canterbury, New Zealand. Int. J. Tuberc. Lung Dis. 2021; 25(12): 990–994. PubMed Abstract \| Publisher Full Text\n\nJakasania A, Shringarpure K, Kapadia D, et al.: “Side effects--part of the package”: a mixed methods approach to study adverse events among patients being programmatically treated for DR-TB in Gujarat, India. BMC Infect. Dis. 2020 Dec 1; 20(1). Publisher Full Text\n\nAusi Y, Santoso P, Sunjaya DK, et al.: Between Curing and Torturing: Burden of Adverse Reaction in Drug-Resistant Tuberculosis Therapy. Patient Preference and Adherence;2021; vol. 15. : 2597–2607. Publisher Full Text\n\nJoseph P, Desai VBR, Mohan NS, et al.: Outcome of standardized treatment for patients with MDR-TB from Tamil Nadu, India. Indian J. Med. Res. 2011; 133(5).\n\nIsaakidis P, Varghese B, Mansoor H, et al.: Adverse events among HIV/MDR-TB co-infected patients receiving antiretroviral and second line anti-TB treatment in Mumbai, India. PLoS One. 2012; 7(7): e40781. PubMed Abstract \| Publisher Full Text\n\nAkshata JS, Chakrabarthy A, Swapna R, et al.: Adverse Drug Reactions in Management of Multi Drug Resistant Tuberculosis, in Tertiary Chest Institute. J. Tuberc. Res. 2015; 03(02): 27–33. Publisher Full Text\n\nVieira DEO, Gomes M: Adverse effects of tuberculosis treatment: Experience at an outpatient clinic of a teaching hospital in the city of São Paulo, Brazil. J. Bras. Pneumol. 2008; 34(12): 1049–1055. PubMed Abstract \| Publisher Full Text\n\nArbex MA, Varella M d CL, de Siqueira HR , et al.: Antituberculosis drugs: Drug interactions, adverse effects, and use in special situations. Part 1: First-line drugs. J. Bras. Pneumol. 2010; 36.\n\nLan Z, Ahmad N, Baghaei P, et al.: Drug-associated adverse events in the treatment of multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet Respir. Med. 2020 Apr 1; 8(4): 383–394. PubMed Abstract \| Publisher Full Text\n\nPrafulbhai Rupani M, Dave JD, Parmar VB: THE NATIONAL MEDICAL JOURNAL OF INDIA Adverse drug reactions and risk factors for discontinuation of multidrug-resistant tuberculosis regimens in Gujarat, western India.2020.Reference Source\n\nPrasad R, Verma SK, Sahai S, et al.: Efficacy and safety of kanamycin, ethionamide, PAS and cycloserine in multidrug-resistant pulmonary tuberculosis patients. Indian J. Chest Dis. Allied Sci. 2006; 48(3): 183–186. PubMed Abstract\n\nPrasad R, Singh A, Srivastava R, et al.: Frequency of adverse events observed with second-line drugs among patients treated for multidrug-resistant tuberculosis. Indian J. Tuberc. 2016; 63(2): 106–114. PubMed Abstract \| Publisher Full Text\n\nShinde KM, Pore SM, Bapat TR: Adverse reactions to first-line anti-tuberculous agents in hospitalised patients: pattern, causality, severity and risk factors. India. J. Med. specialities. 2012; 4(1). Publisher Full Text\n\nShah H, Yasobant S, Patel J:A Study on Adverse Drug Reaction Among TB Patients.xlsx. figshare. Dataset.2022. Publisher Full Text" }	[ { "id": "235642", "date": "27 Feb 2024", "name": "Gyanshankar Mishra", "expertise": [ "Reviewer Expertise Respiratory diseases", "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\n\" This manuscript provides a thorough analysis of adverse drug reactions (ADRs) associated with first and second line anti-tuberculosis (TB) treatment drugs. It offers valuable insights into the ADRs experienced by patients in the National TB Program of India, making a significant contribution to the existing body of literature. One key finding is the increased incidence of ADRs in patients with drug-resistant TB compared to those with drug-sensitive TB. This observation is crucial for a deeper understanding of the challenges in treating drug-resistant TB. I recommend enhancing the discussion on this point at the end of the first paragraph following the line addressing the variance in ADR prevalence. It would be pertinent to include a statement that emphasizes the doubling of ADRs in patients with drug-resistant TB compared to those with drug-sensitive TB, reflecting the lesser efficacy and increased toxicity of second-line drugs. This insight underscores the necessity for prolonged treatment durations and a higher expectation of ADRs during the course of second-line drug therapy. Additionally, the study's identification of the most common ADRs, including gastric discomfort, arthralgia, and cutaneous reactions, is consistent with findings from other studies in India. In the discussion section following the mention of these ADRs, I suggest incorporating a reference to a significant study involving 750 TB patients who received the daily fixed-dose combination anti-TB treatment under the National TB Elimination Program (NTEP) [1]. This study provides important context on peripheral neuropathy, particularly in drug-resistant TB cases treated with second-line drugs. Emphasizing the importance of early recognition and management of linezolid-associated peripheral neuropathy to prevent irreversible progression would be valuable [2]. Furthermore, the role of arthralgia, especially in relation to pyrazinamide use, warrants additional discussion. Highlighting the importance of serum uric acid estimation in differentiating hyperuricemic from normouricemic arthralgia and the potential role of uric acid-lowering drugs, in addition to standard therapies, in managing anti-TB treatment-induced hyperuricemic arthritis, is crucial for a comprehensive understanding of these ADRs [1]. The inclusion of these points will enhance the depth and comprehensiveness of the discussion in the manuscript, ensuring it provides a detailed and nuanced understanding of the ADRs associated with TB treatment in India. The manuscript, with these additions, will be an invaluable resource for healthcare professionals and researchers in the field of TB treatment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "12040", "date": "23 Jul 2024", "name": "Harsh Shah", "role": "Author Response", "response": "Dear [Reviewer/Editor], The revised manuscript has incorporated the feedback from the peer review process. The suggestions from the mentioned study have been integrated into the discussion sections. One limitation of the study was that drug-specific adverse drug reactions (ADRs) were not established, and general considerations of ADRs were taken into account, particularly in drug-resistant tuberculosis (DR-TB) cases, due to the absence of specific case records. Therefore, linezolid-specific ADRs or those related to second-line drugs were not explored, as the primary intention was to identify general characteristics and contributing factors. However, the feedback received highlights the necessity for further exploration to address the continuum of treatment by creating a system that offers more detailed guidance on ADRs. In the same direction, the present study underscores the need to equip frontline workers and medical officers in health facilities with the knowledge and skills to manage ADRs efficiently. Thank you for your valuable insights and suggestions, which have significantly contributed to improving the manuscript." } ] }, { "id": "301300", "date": "23 Jul 2024", "name": "Aditi Gupta", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKindly elaborate Serious adverse events (SAE) and ADR categories also need to be explained It has been proven that elderly develop more ADR but this is not the case with your study. BMI, age, comorbidity have strong association with ADR. Given tables and text do not support this. Kindly elaborate, how did you reach private patients for interview. Since, there is lot of recall bias, results cannot be generalized. At what time of treatment, did you interview the patient? Time of interview is very important in assessing ADR and decreasing recall bias. Ideally different groups at different time point of treatment must be evaluated or even same group at different time points if feasible. Design of the study makes it very less 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? 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": "12072", "date": "26 Jul 2024", "name": "Harsh Shah", "role": "Author Response", "response": "Dear Reviewer, We request you to approve this article as we have incorporated your suggestions in our manuscript. Kindly do needful for its wider dissemination. Regards" }, { "c_id": "12073", "date": "26 Jul 2024", "name": "Harsh Shah", "role": "Author Response", "response": "Greetings, Reviewer We appreciate your input, however we would like you to review the response that addresses the comments you expressed regarding the manuscript. We believe that the evidence that this article generates will be necessary to improve India's ADR surveillance system as part of the National TB Elimination Program. 1. Kindly elaborate Serious adverse events (SAE) and ADR categories also need to be explained The study had classified ADRs into various categories based on severity, including mild, moderate, and severe, with SAEs defined as those resulting in significant health risks, requiring hospitalization, or leading to long-term consequences. This classification is based on the guidelines and protocols defined in context with the National TB Elimination Program (NTEP). 2. It has been proven that elderly develop more ADR but this is not the case with your study. While literature indicates a higher prevalence of ADRs among elderly patients, the findings may reflect demographic variances or differences in treatment regimens that could mitigate these risks. It is crucial to consider that the elderly in the study may have had different TB comorbidities or medication profiles compared to the general population. The results provide valuable insights into the complexity of ADRs and underscore the need for targeted interventions in vulnerable groups. 3. BMI, age, comorbidity have strong association with ADR. Given tables and text do not support this. While the study did not establish significant correlations in the reported data, it is important to consider that these associations can vary based on geographical and socioeconomic factors affecting the patient population. The findings contribute to the broader understanding of ADRs in TB treatment and highlight the necessity for ongoing research to explore these relationships further, particularly in public health settings where such data can inform resource allocation and management strategies. We made references supporting this evidences, however we believe there should be more in depth operational research or longitudinal case control study will provide more structured findings in alignment of the NTEP in India. 4. Kindly elaborate, how did you reach private patients for interview. We reached out the private patients through the National Tuberculosis Elimination Program (NTEP) staff, project staff and through various stakeholders, to ensure a comprehensive representation of persons with TB across both public and private healthcare sectors. 5. Since, there is lot of recall bias, results cannot be generalized. While we acknowledge that recall bias can affect data accuracy, we have already mentioned and narrated it under the limitation of the study. In order to reduce the recall bias, we had further employed a semi-structured questionnaire designed to mitigate these issues by prompting patients to recall their experiences in the context of their ongoing / completed treatment. We ensured that the interviewed study subjects have the medical records available in Ni-kshay or with treatment report card at health facilities in case of requirement to check bias. 6. At what time of treatment, did you interview the patient? Time of interview is very important in assessing ADR and decreasing recall bias. Ideally different groups at different time point of treatment must be evaluated or even same group at different time points if feasible. The study interviews were conducted from the individuals with TB on post completion / on going of their TB treatment. We have tried to incorporate various representative of patients in the study to understand the occurrence of ADRs at various phase of treatment. We could not make separate groups in absence of budgetary restriction to repeat visits for periodic timeframe. The study design was cross sectional and interview was one time event. This is limitation that we have mentioned into the manuscript. 7. Design of the study makes it very less informative. While the design may appear limited, it was intentionally developed to capture data from a diverse patient population. The findings provide critical insights into ADRs experienced by individuals undergoing TB treatment at point interval (with cross sectional design), which is vital for program policies aimed at improving drug safety and efficacy. The study highlights the importance of monitoring ADRs during various treatment phases, which is essential for developing effective TB interventions that ensure patient safety and optimize treatment outcomes. Kindly do revisit the manuscript and comments that we addressed here. Regards" } ] } ]	1	https://f1000research.com/articles/11-1388
https://f1000research.com/articles/13-495/v1	17 May 24	{ "type": "Review", "title": "A review on the phytochemistry and biological activities of Curculigo latifolia Dryand ex. W.Aiton", "authors": [ "Amanina Yusrina Taufik", "Hartini Mohd Yasin", "Norhayati Ahmad", "Masayoshi Arai", "Fairuzeta Ja'afar", "Amanina Yusrina Taufik", "Hartini Mohd Yasin", "Norhayati Ahmad", "Masayoshi Arai" ], "abstract": "Curculigo latifolia Dryand. ex W. T. Aiton, from the genus Curculigo, is a medicinal plant traditionally used to treat numerous illnesses such as fever, stomach aches, jaundice, wounds, and inflammation. C. latifolia is a perennial herb that is widely found in tropical and subtropical regions, such as Southeast Asia, Southern China, Bangladesh, Australia, and the Andaman Islands. This review collates the reported studies on the different aspects of C. latifolia from its plant description, nutritional value, phytochemistry, chemical composition, and pharmacological properties. This review aims to identify gaps in the literature and provide useful references for future work on this plant. Previous studies have shown that C. latifolia contains high mineral contents of calcium, iron, and magnesium, which are essential components of human health. Moreover, the plant is rich in phytochemicals, which play a prominent role in various pharmacological activities. The most common compounds identified included curculigoside, crassifoside I, nyasicoside, and curculigine. C. latifolia demonstrated high antioxidant activity through its ability to scavenge superoxide anions, 1,1–diphenyl–2–picrylhydrazyl (DPPH) and 2,2’-azino–bis(3–ethylbenzthiazoline–6–sulfonic acid) (ABTS) radicals, reducing ferric ions to ferrous complexes, iron chelation, and B-carotene bleaching. It was also shown that the roots, stems, and leaves of C. latifolia were effective in exerting antimicrobial activity against several microbial strains, including Bacillus cereus, Bacillus subtillis, Enterobacter aerogenes, Erwinia sp., Klebsiella sp., Pseudomonas sp., Candida albicans, Salmonella choleraesuis and Staphylococcus aureus. Moreover, the root, fruit, leaf, petiole, and rhizome extracts were found to improve glucose uptake and insulin secretion in diabetic rats, suggesting their antidiabetic potential. C. latifolia presents a wide range of medicinal properties that could make it a promising functional food or source of food supplements to prevent nutrition–related or chronic diseases.", "keywords": [ "Curculigo latifolia", "antioxidant", "anti–diabetic", "curculin", "medicinal", "functional food" ], "content": "1. Introduction\n\nThe genus Curculigo of the Hypoxidaceae family consists of 20 species of perennial herbs that are typically distributed in tropical and subtropical regions such as India, China, Southeast Asia, and Australia.ome species in this genus are known for their medicinal properties and have often been used in traditional therapeutic remedies.1 Curculigo orchioides is a species native to India and China and is known to have a long–standing tradition of medicinal use, including the treatment of jaundice, limb limpness, knee joints, and diarrhea.2 Additionally, the rhizomes of C. orchioides have been suggested to be favorable for maintaining the health of the liver and kidneys.3 Curculigo species, which are widely known for their medicinal properties and a variety of health benefits, are called Curculigo capitulata. This plant is generally found in China, South Asia, Southeast Asia, Australia, Taiwan, and the Pacific Islands, and is traditionally used for the treatment of chronic bronchitis, nephritis, hemorrhoids, gonorrhea, and asthma.2,4 In Africa, an indigenous species of Curculigo, known as Curculigo pilosa is recognized as a useful remedy for infertility, leukemia, coughs, diabetes, genital infections, and sterility.2,5\n\nFurthermore, previous studies have reported that these Curculigo species are composed of phytochemical constituents, such as phenols, phenolic glycosides, norlignans, and terpenoids, which are known to promote various biological activities.2 Many studies have revealed that these compounds have anti–osteoporotic,6 nephroprotective,7 antioxidant,8 antibacterial,9 anti-diabetic,10 and anti-inflammatory activities.11\n\nCurculigo latifolia Dryand. ex W. T. Aiton is a Curculigo species found in Brunei Darussalam, and is more recognizable by its local name, Lemba. This plant is also synonymous with Molineria latifolia, Curculigo villosa and Aurota.12 In Malaysia, the species is known as Kelapa Puyuh, Lumbah Puyuh, Pinang Puyuh, and Nyiur Lember, whereas in Indonesia, it is identified as Marasi, Keliangau, Prakuwang, Lumpa, Doyo, and Kehoang.13 Additionally, in Thailand, C. latifolia is known as Chaa Laan, Ma Phraao Nok Khum and Phraa Nok, whereas in Vietnam, it is called Cồ Nốc Lá Rộng, Sâm Cau Lá Rộng.13\n\nC. latifolia is widely distributed in most Southeast Asian regions, such as Myanmar, the Philippines, Thailand, Vietnam, and Cambodia, as well as Borneo Island, which includes Malaysia, Brunei, and Indonesia.13 This plant has also been reported in Southern China, Bangladesh, Australia, and Andaman Islands.14 Lim13 suggested that this species of Curculigo is predominantly susceptible to growth in warm temperate regions and humid tropical and subtropical areas. Moreover, the plant is mostly widespread on slopes and forests, as well as in highland areas with an altitude of 1500 – 2000 m.15 A previous study has investigated the effects of light and soil media on the growth of C. latifolia.16 In the present study, the plant showed the most prominent growth progression at 50% light intensity, indicating that C. latifolia generally prefers moderate shading.16 The plant was also found to grow in the wild in fertile, well–drained soils rich in organic matter. Additionally, the study suggested that soil media comprising topsoil, organic manure, and sand in a ratio of 2:3:1 showed the best results in terms of plant growth.16\n\nC. latifolia is claimed to have medicinal value in the treatment of asthma, hemorrhoids, jaundice, skin disease, and diabetes.17 Moreover, several studies on C. latifolia have shown the presence of phenols and flavonoids that could counteract free radicals and thus assist in a broad spectrum of pharmacological activities, such as anti–diabetic, antioxidant, and antimicrobial activities.10,18,19 Each part of the Curculigo latifolia plant plays a role in promoting certain biological activities. In traditional medicine, the rhizome of C. latifolia is used to treat jaundice, menorrhagia, and fever.18,20 It can also be boiled with Areca catechu and Hibiscus rosa–sinensis which helps in regulating menstrual bleeding and healing ophthalmia.13 Moreover, the flowers and roots have been used for treating stomach or urinary disorders, and in Malaysia, the leaves and roots are usually used for inflammation and wounds.18,21 In Brunei Darussalam, the root is utilized to cure headaches and mouth thrush, while the mixture of roots and rhizomes can help with diarrhea.22 Additionally, traditional Thai medicine claims that the root can be used as a tonic to regulate blood circulation.23 It is also known to have several other medicinal properties, such as hemorrhoids and asthma.24 Additionally, previous findings have also suggested that the phytochemicals found in C. latifolia exhibit hepatitis B virus inhibitor, anti–diabetic, anti–arthritic, anti–tumor, anti-inflammatory, estrogenic, and sexual behavior-modifying activities.18,25\n\nAdditionally, C. latifolia has also been shown to be used for taste modifying activities and as an alternative to low–calorie sweeteners owing to its composition of sweet proteins known as curculin or neoculin.26 This protein is isolated from the fruits of the C. latifolia plant and can produce a strong sweet taste when mixed with an acidic solution.27 It was found that curculin in its heterodimeric form (also known as neoculin) can interact with T1R2–T1R3 sweet–taste receptors, which resembles the sweettasting activity of sugar and aspartame.28 Additionally, the large cluster found in the basic subunit (NBS) of the sweet proteins, which is composed of six basic residues on its surface, was suggested to contribute to the sweetness and taste–modifying activities.29\n\nIn addition, C. latifolia has other practical uses: the sturdy, durable, and lightweight fibers of its leaves have been utilized for making fishing nets, strings, and food wrappings.30 It was also revealed that the fibers showed characteristics similar to those of cotton fibers, making them an alternative material in clothing production. In Eastern Kalimantan, C. latifolia fibers have traditionally been used for weaving fabrics and handbags.31,32 Additionally, the plant was considered for its potential as a natural dye, and Shaari30 found that the leaves and flowers produced promising colors ranging from dark green to yellow, which can act as dyes.\n\nThis review presents several reported findings that demonstrate the different aspects of C. latifolia such as its morphology, nutritional value, phytochemistry, chemical composition, and biological activity. To the best of our knowledge, there are limited publications on this plant, and no reported paper has summarized the studies carried out. The aim of this study was to determine the gaps in the literature and provide baseline information that may be useful for future studies on this plant.\n\n\n2. Method\n\nScientific literature was collected using web search engines, including Scopus, PubMed, Science Direct, Google Scholar, and Taylor and Francis, from inception until August 2023. The search was conducted using keywords such as ‘Curculigo latifolia’, ‘Molineria latifolia’, ‘Curculigo species’,’ Curculigo latifolia OR Molineria latifolia’,’ C. latifolia AND phytochemistry, “’C. latifolia AND pharmacological activities’, and”’C. latifolia medicinal values’. Online resources such as botanical websites were also considered to determine the geographic distribution, description, and synonyms of the plant. The author attempted to document the relevant literature that primarily focused on the phytochemistry and biological activities of C. latifolia.\n\n\n3. Morphology of Curculigo latifolia\n\nCurculigo latifolia is a perennial herbaceous monocotyledon that has an erect rhizome of about 9.5 cm long and forms a cluster of green leaves up to 1 m tall, as seen in Figure 1.33 The leaves of this plant are generally thin, broad, and elliptical, making them suitable for use as wrappings, fishing nets, and strings.30 They are also mainly subglabrous or pubescent and have entire margins, followed by an acuminate tip.26,31 Additionally, they commonly have leafstalks that are approximately one–third of the length of the leaves, which are generally 30 – 100 cm long and 5 – 10 cm wide.26 The inflorescences are compact and ovoid or cylindrical, with a length of 1 – 6 cm. The flowers are yellow and have lanceolate, hairy bracts and stamens of 1.5 cm long that are slightly shorter than the slender style.13 The fruits of C. latifolia are white to green and have been found to be 10 – 25 mm wide and sweet. They are usually ovoid and long-beaked, with small black seeds.13,33\n\n(a) whole plant, (b) leaves, (c) petiole/stem, (d) roots, (e) callus, (f) plantlet leaves, (g) fruits, (h) flowers, (I) rhizomes (Images retrieved from Babaei et al. (2014),34 Haryanto et al. (2023),35 Nur, Setiawan, et al. (2023a),36 Umar et al. (2023),37 Umar et al. (2021a)38, and Umar et al. (2021b)39).\n\n\n4. Nutritional value\n\nC. latifolia fruits have been reported to contain various nutritional content such as 1.08 mg/100 g of fructose, 1.08 mg/100 g sucrose, 5.61 mg/100 g vitamin C and 0.41 mg/100 g potassium (Table 1).4 These mineral nutrients are crucial for the development of strong bones and teeth, management of body fluids, and transmission of nerve signals, all of which are essential for sustaining optimal functions of the body.40\n\nTheir magnesium, calcium, and iron contents were shown to be abundant compared to other fruits. For instance, the magnesium content of C. latifolia (117.6 mg/100 g)4 was richer than figs (67.90 mg/100 g)41 and banana (29.39 mg/100 g).42 Magnesium is an important component in the formation of proteins, muscle function, and immune system support, and low levels of magnesium can lead to diabetes, cardiovascular disease, and neurological disorders.40,43 This shows C. latifolia fruits contain a desirable amount of magnesium that could potentially serve as a supplement for improving overall human health, specifically in areas where magnesium deficiency is prevalent.\n\nFurthermore, C. latifolia fruits (3452.01 mg/100 g)4 exhibited higher calcium content compared to figs (154.55 mg/100 g)41 and mulberry (1493.22 mg/100 g),44 which enhances their suitability for maintaining strong bones, supporting muscle and nerve function, and regulation blood pressure.45 Calcium is one of the micronutrients essential to the human body to maintain optimal health, and the recommended dietary intake of calcium ranges from 1000 to 1300 mg/day.46,47 The calcium content of C. latifolia fruits could support individuals with insufficient calcium levels, subsequently mitigating the chances of osteoporosis, hypertension, and cardiac arrhythmias.45\n\nIron is another major mineral in the body that plays an important role in metabolic functions, such as deoxyribonucleic acid (DNA) synthesis, oxygen, and electron transport.48 The iron content of C. latifolia was 872. 93 mg/100 g,4 which is higher than dried apricots at 6.97 mg/100 g dried weight basis,49 and Tunisian dates at 7.2 mg/100 g.50 Generally, the daily iron intake from foods and supplements is recommended to be 19.7–20.5 mg/day for men and 17.0–18.9 mg/day for women aged above19 years old.51 Excessive amounts of iron can generate oxygen free radicals, which can lead to tissue damage and organ failure.52 Therefore, maintaining a balanced consumption of minerals is important for the proper functioning of organs, metabolic and homeostatic processes in the body, and protection against illnesses such as cancer, diabetes, and heart disease.53\n\nThe above literature suggests C. latifolia fruit is a viable food source for providing essential nutrients, making it a potential ingredient in functional food applications. However, very few studies have reported the mineral content of C. latifolia fruits and plants as a whole. Therefore, more investigations, such as fatty acid profile, vitamin analysis, amino acid analysis, and clinical trials, are needed to provide more information about its nutritional value.\n\nMoreover, the relatively low sugar content could potentially make it suitable for low–calorie diets, which are suggested for obese and diabetic patients.54 Further studies exploring the glycemic index and cholesterol levels of this plant could provide an understanding of the blood sugar response and overall effects on the body. Nevertheless, C. latifolia fruits have promising nutritional content and can be a valuable supplement for a healthy body.\n\n\n5. Phytochemistry\n\nPrevious studies have revealed the phytochemicals detected in different parts of C. latifolia (Table 2). Lumbangaol55 found that the fruits of this plant contain alkaloids, tannins, saponins, and flavonoids. This study also observed a tannin concentration of 35.6 ppm in the fruit, potentially contributing to a range of pharmacological activities. Akkarasiritharattana and Chamutpong18 also showed similar results for the underground and aerial parts of C. latifolia, detecting the presence of flavonoids, tannins, steroids, terpenoids, alkaloids, and glycosides using high-performance thin-layer chromatography (HPTLC). Other findings by Umar et al.56 and Umar et al.37 examined the calluses, plantlet leaves, rhizomes, leaves, and petioles of C. latifolia and found that these parts contain alkaloids, norlignan, phenolic glycosides, steroids, and terpenoids. Research has indicated that these constituents are responsible for numerous biological activities, such as antioxidant and anti-inflammatory activities, which are valuable properties for the production of functional foods.57 Additionally, these bioactive compounds generally exert a significant positive impact on human health and bodily functions while also regulating, preventing, and reducing the severity of chronic diseases.57 However, systematic investigations of these isolated compounds and the biological effects of C. latifolia should be conducted to validate these findings further.\n\n(+) denotes the presence of phytochemicals, while (–) indicates that the absence of phytochemical and (NR) suggests that the presence of phytochemical was not reported.\n\n\n6. Chemical composition\n\nA literature search of the chemical composition of C. latifolia yielded various studies that showed presence of different chemical groups depending on the different parts of the plant, where Table 3 outlines some of the common compounds identified. C. latifolia roots, fruits, rhizomes, leaves, petioles, calluses, and plantlet leaves contain constituents from different classes of secondary metabolites, such as phenolics, glycosides, flavonoids, flavones, triterpene lignans, cycloartane, alkaloids, sisterols, and proteins. These active compounds play a key role in the interaction with other molecules, receptors, enzymes, or proteins in the body, leading to various health benefits, such as improvements in digestion and cardiovascular health, and a reduction in the risk of chronic diseases.57 Therefore, C. latifolia is a potential source of functional food ingredients owing to its health–promoting bioactive compounds. In the next section (Section 7. Biological activities) elaborates on the biological activities that have been carried out on different parts of C. latifolia which could be associated with their active compounds.\n\nZabidi et al.58 identified the chemical compounds in the root extract of C. latifolia using liquid chromatography–mass spectrometry (LC–MS) analysis, which was found to contain phenolic compounds such as monobenzone, phloridzin, mundulone, scandenin, pomiferin, dimethyl caffeic acid, hordatine A, ubiquinone, hydroquinone, frangulin B, rubratoxin B, and emmotin A. These phytochemicals have a diverse range of bioactive attributes that can play a significant role in exerting pharmacological activities such as anti–diabetic, antimicrobial, anti–inflammatory, antioxidant, and anti–cancer.59 For instance, hydroquinone is known to exhibit antioxidant activity by preventing oxidative damage in human cells,60 whereas ubiquinone plays a key role in cellular metabolism and protection against lipid peroxidation.61\n\nFurthermore, Maliwong et al.23 used high-resolution electrospray mass spectrometry (HR–ESI–MS), which revealed phenolic glycosides, such as molineriosides A–C, curculigoside, curculigoside H, 3–hydroxy–5– methylphenyl 1–O–β–D–glucopyranosyl–(1→6)–β–D–glucopyranoside, nyasicoside, crassifoside B, 1–O–methyl– curculigine, 1–O–methyisolcurculigine, capituloside, and curcapicycloside. Curculigoside is reported as one of the major bioactive compounds in another Curculigo species, Curculigo orchioides, where it was found to promote neuroprotection, anti–arthritic, anti–osteoporosis, and anti–tumor activities.2 Curculigoside has also been suggested to exert significant antioxidant activity, which ameliorates learning performance and bone loss in mutated transgenic mice with Alzheimer’s disease.20\n\nA more recent study by Nur et al.62 reported the presence of methyl–3–hydroxy–4–methoxybenzoate, sugiol (diterpene), stigmastan–3,6–dione (steroid), aviprin (flavonoid), lucialdehyde B (sesquiterpene), guaiacol (phenol), and smilaxin (saponin). Avipirin is a bioactive compound that exhibits antibacterial, antifungal, and phytotoxic activities.63 Guaiacol has been reported to effectively inhibit human carbonic anhydrase isoenzymes,64 and the compound smilaxin has demonstrated immunostimulatory, antiproliferative, and human immunodeficiency virus (HIV) –1–reverse transcriptase inhibitory activities.65\n\nCurculin, a sweet protein, is found in the fruit extract of C. latifolia. This protein has sweet–tasting and taste–modifying properties that can enhance the sweetness of acidic or tasteless substances.26 Moreover, its sweet properties make it a promising option for low–calorie sweeteners, which may be beneficial for individuals aiming to reduce their sugar or calorie consumption.34\n\nMoreover, several phytochemicals such as berberine, hordatine A, robustine (alkaloids), frangulin B (anthraquinone), pomiferin (flavonoid), and monobenzone (aromatic hydrocarbons) have been identified.10 These chemical compounds generally exhibit pharmacological effects. For example, berberine and frangulin B have anti–inflammatory properties,66,67 pomiferin has high antioxidant activities,68 and monobenzone can inhibit the growth of acute myeloid leukemia.69\n\nThe edible fruit of C. Latifolia could be a promising functional food that can provide health–promoting and taste–enhancing effects owing to the presence of phytochemicals and sweet proteins. However, there are still limited studies on the unique compounds of C. latifolia and curculin; therefore, further investigations into its glycemic index, cholesterol level, and anti-diabetic, anti-inflammatory, and anti–cancer activities will provide more information about it.\n\nIn a previous study by Ooi et al.,20 the phenolic composition of the rhizome extract of C. latifolia was determined using high-performance liquid chromatography with diode–array detection (HPLC–DAD). Several active components have been identified, including cinnamic acid, curculigoside, syringic acid, ferulic acid, and protocatechuic acid. The findings also highlighted the efficacy of ethyl acetate solvent in extracting a higher amount of curculigoside and cinnamic acid compared to methanolic rhizome extract.20\n\nIn recent studies by Umar et al.56 and Umar et al.,37 the chemical composition of the ethanolic rhizome extract of C. latifolia was determined using ultra-high-performance liquid chromatography–Q Exactive hybrid quadrupole–Orbitrap high-resolution accurate mass spectrometry (UHPLC–Q–Orbitrap HRMS). This sensitive and accurate method detected the presence of active components such as lycorine, vanillin, nyasicoside, 4–hydroxy–phenol, and curculigoside B.39,56 Additionally, the study also identified some isolates that were previously extracted from other Curculigo species such as orchioside B, orcinol glycoside, curculigine C, curculigosaponin G and C, from Curculigo orchiosides,1,70 capituloside and crassifoside I from Curculigo capitulata,71,72 and (1S,2R)–O–methylnyasicoside from Curculigo sinensis.73\n\nMad Nasir et al.74 also found Curculigo isolates in methanolic rhizome extracts using ultra–high performance liquid chromatography mass spectrometry (UHPLC–MS). These included curculigine, curculigine M and G, curcupicycloside, sinensigenin A, 1–O–methylisocurculigine, brevicaside B, crassifoside C and D, curculigenin, sinenside B, and orchioside J.74\n\nC. latifolia is rich in chemical diversity, some of which can potentially exert various physiological effects. Some of the isolates of Curculigo species that have been reported for their promising biological effects include orcinol glucoside and crassifoside H, which improve depressive behaviors (antidepressant–like effects) in chronic unpredictable mild stress rat models.75,76 However, many of these compounds have not yet been fully investigated for their biological activity. Therefore, extensive studies on these unique isolates are needed to understand the mechanisms underlying their biological activities, such as antioxidant, anti–diabetic, and antimicrobial activities.\n\nUmar et al.56 detected chemical components in the leaves of C. latifolia, where some of the compounds were similar to those found in the rhizome extract. This study identified nyasicoside, vanillin, lycorine, orcinol glycoside, crassifogenin A, behenic acid, pothobanoside C, daucosterol, stigmasterol, curculigine C, curculigosaponin C, E, G, H, and I; crassifoside C, E, and I; curculigoside B and C; and orchioside A and B.39,56,74 Some of these compounds have been reported to exhibit anti-estrogenic and anti-allergic activities against estrogen–responsive human breast cancer cell lines.77 Plant–derived sterols, such as daucosterol and stigmasterol, effectively inhibit cell proliferation and reduce tumor size in patients with prostate, colorectal, and breast cancers.78 Additionally, behenic acid showed significant antibacterial activity against Agrobacterium tumefaciens T–37 and Erwinia carotovora EC–1.79\n\nFurthermore, in a study by Mad Nasir et al.,74 numerous compounds were also detected in the methanolic leaf extract, including orcinoside E, orcinol gentiobioside, sinensigenin B, curculigine M, and (Z)-resveratrol 3,4′-diglucoside. Most of these compounds are derived from glycosides and stilbene groups; however, few studies have investigated their biological effects. Generally, glycosides possess a wide variety of biological attributes, such as anti–inflammatory, antiseptic, anti-rheumatic, and analgesic effects,80 whereas stilbenes exhibit cardioprotective, neuroprotective, anti–diabetic, and cancer treatment and prevention activities.81\n\nPhytochemical screening using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) by Nur et al.62 identified compounds including digiprolactone, 3–tert–butyl–4–methoxyphenol, axedarachin C and 4–O–caffeoylquinic acid–1, and quercetin. Quercetin is regarded as one of the most effective antioxidants that can scavenge free radicals and mitigate diseases associated with oxidative stress such as diabetes, cancer, allergies, inflammation, and gastrointestinal and cardiovascular diseases.82\n\nUmar et al.37 identified the chemical compounds in C. latifolia callus and plantlet leaves, which consist of ecdysterone, a natural anabolic agent that can inhibit the proliferation of breast cancer cells,83 and emodin dianthrone,37 which possesses antidiabetic properties.84 Moreover, the petiole, callus, and plantlet leaves showed the presence of breviscapin, a bioactive compound that was previously extracted from Erigeron breviscapus with the ability to enhance cerebral blood flow and microcirculation and resist platelet aggregation.85 C. latifolia callus also contained the compound salidroside, which has effective properties in ameliorating memory and emotional behavior in adult mice,37,86 and theanaphthoquinone, which has been shown to induce cell death in breast cancer cells.37,87\n\nThese bioactive compounds are important for managing various diseases and should be included in the human diet because of their ability to provide energy and nutrients and contribute to overall well–being.57 Therefore, it is worthwhile to further study the bioactivities of the isolated chemical compounds of C. latifolia to validate the findings for potential use in pharmaceuticals or food supplements.\n\n\n7. Biological activities\n\nC. latifolia has been involved in several in vitro and in vivo investigations, where the plant was shown to exhibit bioactivities such as antioxidant, antimicrobial, anti–diabetic, sperm quality, cytotoxicity, anti–aging and ultraviolet protection activities (summarised in Table 4). The diverse range of bioactivities of plants may be attributed to their high content of chemical compounds. An overview of the biological evaluations carried out on C. latifolia is described in this section.\n\nDPPH refers to 1,1–diphenyl–2–picrylhydrazyl, ABTS is 2,2’–azino–bis(3–ethylbenzthiazoline–6–sulphonic acid radical cation scavenging, FRAP is ferric reducing antioxidant power, SOD is superoxide dismutase, and NR indicates that the information was not reported.\n\nDifferent types of assays have been employed to investigate the antioxidant activity of C. latifolia, and each assay generally involves different chemical reactions and mechanisms.88 Some common antioxidant assays that were involved can be seen in Table 5, which include 1,1–diphenyl–2–picrylhydrazyl (DPPH), 2,2’-azino–bis(3–ethylbenzthiazoline–6–sulfonic acid) (ABTS) radical cation scavenging, and ferric reducing antioxidant power (FRAP) assays. In a previous study by Ooi et al.,20 it was reported that C. latifolia rhizome extract exhibited promising scavenging activity for both DPPH and ABTS radicals, with a Trolox–comparable capability. This study suggested that these antioxidant activities were significantly correlated with the bioactive compounds in the extract, such as phenolics and flavonoids.20 The unique features of these compounds, such as their functional groups, configuration, substitution, and amount of hydroxyl groups, essentially facilitate protection against oxidative damage by radical neutralization, iron binding, and reducing power capacities.89 The report by Ishak and Zabidi24 is consistent with Ooi et al.,20 where high contents of flavonoids and phenolics in the extracts also corresponded to antioxidant activities. The study demonstrated that the root extract of C. latifolia with high phenolic and flavonoid contents under subcritical water extraction showed DPPH radical scavenging activities of 128.70 mg Trolox equivalents/g sample and ABTS scavenging activity of 66.78 mg Trolox equivalents/g sample.24\n\n(DPPH (1,1–diphenyl–2–picrylhydrazyl), ABTS (2,2’–azino–bis(3–ethylbenzthiazoline–6–sulphonic acid) radical cation scavenging, FRAP (ferric reducing antioxidant power), and SOD (superoxide dismutase)).\n\nMoreover, in the findings of Nur et al.,62 different extracts of C. latifolia roots, stems, and leaves reported varying antioxidant capacities, in ABTS study, ethanol and ethyl acetate extracts of C. latifolia exerted the most significant activity by contributing proton radicals to free radical compounds. Meanwhile, β-carotene bleaching assay demonstrated that ethyl acetate extracts of C. latifolia roots, stems, and leaves could effectively prevent β-carotene decomposition upon oxidation of linoleic acid to hydroperoxide compared to the other extracts. In the FRAP assay, the root extracts (ethyl acetate and ethanol) showed the strongest antioxidant activity for reducing Fe3+ to Fe2+ complexes. Nur et al.62 suggested that the hydroxyl groups in the phenolic compounds in the extracts were highly effective in the chelation of iron and subsequently reduced it via a redox reaction.\n\nA recent study by Umar et al.56 also suggested that the rhizome extract of C. latifolia showed higher antioxidant activity than the leaf and petiole extracts. In this study, the active components that significantly contributed to the antioxidant activity were evaluated using Fourier transform infrared (FTIR) spectroscopy and UHPLC–Q–Orbitrap HRMS combined with partial least squares (PLS) assay. Based on this analysis, a chemical compound identified as unknown (185) with chemical formula C47H59O7 was found to be a major contributor to the activity, whereas phenolics (curculigoside B, 2,4– dichloro–5–methoxy–3–methylphenol, orchioside B), cycloartane triterpene (curculigosaponin G), and norlignan compounds (1,1–bis (3,4–dihydroxyphenyl)–1–(2–furan)–methane and (1S,2R)–O–methylnyasicoside) showed only a minor contribution.62 Moreover, the presence of phenolics, norlignan, and alkaloid compounds, such as vanillin, crassifoside A, and lycorine, in C. latifolia extracts exhibited antioxidant properties.39 Regarding the functional groups, Umar et al.56 determined that compounds with –OH, C=O, C–O, –C–H, C–C, and C–OH groups strongly influenced the antioxidant capacity. This result agrees with the study by Mad Nasir et al.,74 which suggested a positive correlation between phenolics and DPPH antioxidant activity of C. latifolia extracts. The report showed that the rhizome extract produced a high DPPH radical scavenging activity with IC50 value at 18.10 ± 0.91 μg/mL which is closely comparable to that of its standard ascorbic acid (IC50 11.49 ± 0.071 μg/mL). In contrast, the fruit and leaf extracts displayed a lower antioxidant activity against DPPH assay, with IC50 values of 26.99 ± 1.58 and 547.29 ± 5.09 μg/mL respectively.74\n\nIn addition, an interesting finding by Farzinebrahimi et al.15 indicated that the free radical scavenging abilities of tuber and leaf extracts of C. latifolia reached 80% and 60%, respectively. The same study also evaluated a considerable antioxidant activity of 70% and 65% in the callus of tuber and leaf extracts, respectively, suggesting that the plant can be considered to have antioxidant effects.15 Akkarasiritharattana and Chamutpong18 found that the aqueous extract of the underground part of C. latifolia, assessed using DPPH and FRAP assays, also showed favorable radical scavenging activity and reducing power ability. However, the ethyl acetate extract of C. latifolia underground part of C. latifolia in the same study resulted in a lower potential. This may be due to the discrepancy in the solvents used, where more polar phenolic and flavonoid compounds possessing antioxidant properties may have been extracted in greater quantities using water than ethyl acetate.18\n\nWith its high antioxidant activity comparable to ascorbic acid, C. latifolia effectively scavenges free radicals, chelates metal iron, and reduces ferric (III) iron to ferrous (II) iron. The antioxidant properties of the plant can protect cells and tissues in the human body from oxidative stress, which is linked to chronic diseases such as diabetes, cancer, and heart disease.90 This makes C. latifolia a valuable source of functional foods that can help supplement the daily diet with antioxidants, subsequently preventing the body from harmful free radicals. Nonetheless, investigations of the antioxidant effects of different extracts of various parts of the plant and isolated components are still warranted. Further detailed studies are needed to explore the bioactivities of C. latifolia which will support its medicinal value.\n\nPrevious studies have reported that different parts of C. latifolia such as the root, stem, and leaf, exert antimicrobial activities against seven different bacterial strains, including Bacillus cereus, Bacillus subtillis, Enterobacter aerogenes, Erwinia sp., Klebsiella sp., Pseudomonas sp. and Staphylococcus aureus.19 It was demonstrated that by increasing the concentration of all the extracts, with the highest concentration being 100 mg/mL, the elimination and restraint of the microorganisms were gradually more effective.19 A similar observation was made by Lim and Ibrahim91 where the root extract was seen to have the ability to inhibit the growth of Candida albicans, with the lowest minimum inhibitory concentration of the extract being 1.56 mg/mL. In another study, the leaves and tubers from intact C. latifolia plant (in vitro) and callus (in vivo) extracts showed considerable antibacterial activity against Pseudomonas aeruginosa and Klebsiella sp.15 However, the most prominent activity was observed in the tuber extract, which was suggested to contain phytochemicals with antibiotic properties.15 Furthermore, a study by Mad Nasir et al.74 revealed that the methanolic leaf extract had promising antibacterial properties against Staphylococcus aureus (gram-positive bacteria) and Salmonella choleraesuis (gram-negative bacteria), with inhibition zones of ±15.33 mm and ±8 mm, respectively. The active phenolic compounds, such as tetrahydromethylmononya– sine, (2R,4S,5S,6R)–2–ethyl–6–(4–methylphenoxy)oxane–3,4,5–triol, and (Z)–resveratrol 3,4′-diglucoside, in the leaf extracts were found to predominantly react with the enzymes and proteins of the microbial cell membrane.15\n\nGiven the antibacterial effects of this plant, additional investigations, such as in–depth research on the chemical constituents of C. latifolia responsible for its antimicrobial activity, are needed. Additionally, since there are limited studies related to the antimicrobial activities of this plant and its different parts, further detailed investigations are required.\n\nThe antidiabetic properties of C. latifolia have been investigated in multiple studies, both through chemical assays and animal model experiments. In a report by Zabidi et al.10, C. latifolia root extract was found to exhibit a considerably significant inhibition of α– glucosidase and dipeptidyl peptidase–4 (DPP (IV)) enzymes, where its percentage inhibition was in close proximity to that of acarbose, a marketed anti–diabetic drug. In addition, it was found that through the synergistic interaction of phytochemicals in the extracts, such as berberine, flavonoid glycoside (phlorizin), isoflavonoid (mundulone and scandenin), and cinnamic acid derivative (dimethyl caffeic acid), insulin secretion and glucose uptake activity were greatly enhanced.15 The study also suggested that the fruit extract of C. latifolia demonstrated antidiabetic activity, although it was less effective than the root extract. This was possibly because some bioactive compounds in the root extract were absent from the fruit extract, which lowered the efficacy level where fewer synergistic interactions occurred.15 A similar result was also seen in the study by Ishak and Zabidi,24 where the fruit and root extracts displayed promising antidiabetic effects. However, in this study, the leaf extract of C. latifolia showed a lower potential in activity, which may be due to the lack of some active components.\n\nFurthermore, a study by Umar et al.56 found that the rhizome extract exhibited a more significant α– glucosidase inhibition than the leaf and petiole extracts. The study suggested that the isolated compound, unknown–85 (C42H51O6), primarily influenced inhibition, whereas phenolics (orcinol glucoside), cycloartane triterpene (curculigosaponin G, H, and I), and norlignan compounds (1,1–bis (3,4–dihydroxyphenyl)–1–(2–furan)–methane) contributed slightly. Moreover, the fruit of the plant contains a sweet protein known as curculin.34 This protein was suggested to be 500 to 9000 times sweeter than sucrose by weight, and is known for its use as a sugar substitute and taste modifier. Curculin is also recognized as a low–calorie sweetener; thus, it is considered to have a potential in having antidiabetic properties.34 Despite this, there have been no studies to confirm this postulation; therefore, further studies are needed to investigate the glucose uptake, insulin secretion, and α–glucosidase and α–amylase inhibitory effects of curculin.\n\nMoreover, in an in vivo study, C. latifolia fruit:root extract at a 1:1 ratio was found to decrease glucose and lipid levels, as well as increase insulin and adiponectin levels in streptozotocin (STZ)–induced diabetic rats.92 The extract demonstrated its efficiency in inhibiting the disruption of pancreatic β–cells, which is usually induced by STZ in diabetic rats. This indicates that the fruit and root extract of this plant was capable of scavenging radicals that could cause oxidative stress in cells, suggesting its antioxidant properties.92 Another previous study reported the potential for diabetes management linked to an isolated compound from the rhizome extract of C. latifolia known as curculigoside.93 In response to this, it was observed that glucose uptake was improved by the increased availability of glucose transporter type 4 (GLUT4) at the plasma membrane of differentiated 3t3–L1 adipocytes in male Sprague–Dawley rats.93\n\nBased on the reported improvements in glucose uptake, insulin secretion, and the significant α–glucosidase and α–amylase inhibition effects, it can be deduced that C. latifolia possesses promising anti–diabetic properties. Despite this, the reported studies carried out so far revealed that only aqueous and ethyl acetate extracts of the roots, rhizomes, fruits, and leaves were used. Therefore, various other plant extracts can also be considered for the investigation of their anti–diabetic properties.\n\nOoi et al.94 investigated the lipid profile of C. latifolia rhizome extract and reported improvements in total cholesterol levels and increased high–density lipoprotein (HDL) levels in obese diabetic rats. Additionally, the lipoprotein cholesterol ratio of HDL and low–density lipoprotein (LDL) (HDL:LDL) was ameliorated by treatment with 200 mg/kg body weight rhizome extract. This indicated that the extract could potentially help reduce the risk of cardiovascular disease, especially in obese patients.94 Similarly, Ishak et al.92 found that treatment with 50, 100, and 200 mg/kg body weight of fruit improved the lipid profile of obese diabetic rats. The findings demonstrated a significant decrease in total cholesterol, triglycerides, and LDL and an increase in HDL. These findings suggest that C. latifolia is a promising plant for controlling cholesterol levels in the body, which is important for reducing the risk of heart disease and stroke, which are more prevalent in individuals with obesity.\n\nC. latifolia root and leaf extracts have also been demonstrated to improve sperm quality in mice (Mus musculus). According to a study by Jaafar et al.,95 mice fed with C. latifolia root extract showed a greater increase in sperm motility when compared to the control group, whereas the mice fed with C. latifolia leaf extract showed a higher sperm count and viability.\n\nIn addition, a cytotoxicity test was carried out on the root extract of this plant, and Lim and Ibrahim91 suggested that the extract demonstrated no toxicity against brine shrimp eggs, with a lethal concentration (LC50) of 2.25 mg/mL. This finding indicated that the extract had a minimal negative impact on living cells and was less likely to cause a reduction in cell viability or cell death.96 Moreover, a cell viability assessment, which measures the proportion of healthy functional cells, was conducted by Zabidi et al.10 via the MTT (3-[4,5–dimethylthiazol–2–yl] –2,5 diphenyl tetrazolium bromide) assay. The study showed the ability of 3T3–L1 cells to survive at IC20 (153.21 ± 9.65 μg/ml) and IC50 (561.42 ± 6.22 μg/ml) of C. latifolia root extract and IC20 (295.67 ± 7.43μg/ml) and IC50 (495.67 ± 11.31 μg/ml) of fruits extract. Meanwhile, MTT assay by Ooi et al.93 found the IC20 and IC50 values for ethyl acetate fraction of rhizome extract were 111.73 ± 9.57 and 509.59 ± 49.75 mg/mL, respectively. These findings suggest that C. latifolia does not have toxic effects on cells, suggesting that it is safe for use or consumption.\n\nPrevious reports have suggested that C. latifolia compounds have anti–aging and ultraviolet protection properties. An in silico anti–elastase study was conducted by Nur et al.62 through molecular docking to predict the interactions between ligands (C. latifolia compounds) and the elastase target protein (1B0F). From the study, sugiol, aviprin, 4–O–caffeoylquinic acid–1, quercetin and 5,7,3,5–tetrahydroxyflavanone compounds of C. latifolia demonstrated the best interaction against the target protein, where the binding free energies of the compounds were <–6 kcal/mol. This indicated that the compounds of the plant have promising potential to exert anti–elastase activity, which could promote anti–aging effects. In another study by Nur et al.,97 C. latifolia compounds, pomiferin, scandenin, mundulon, and rubratoxin were found to have a negative binding affinity energy against matrix metalloproteinases, MMP–1 (collagenase) and MMP–9 (gelatinase), while 1,1,6–trimethyl–1,2–dihydronaphthalene, orcinol glucoside, and sugiol compounds effectively inhibited the target proteins hyaluronidase. The ability to inhibit target proteins is mainly attributed to the functional groups in the chemical structures of the active compounds, which can readily interact with the amino acid residues of the target proteins.97\n\nFurthermore, Nur et al.98 revealed that hexane, ethyl acetate, and ethanol extracts of Curculigo latifolia leaves, roots, and stems have promising bioactivity in ultraviolet protection against UVA and UVB rays. The study found that The hexane leaf extract demonstrated the highest intensity in absorbing UVA and UVB radiation, with absorption values of 1.192 and 1.804, respectively. Additionally, it was also indicated that leaf hexane, root ethyl acetate, stem ethyl acetate and leaf ethyl acetate extracts at concentration 250 ppm showed the most prominent ultra–protective effect with sun protection factor (SPF) values of 23.65, 16.5, 22.5 and 23.03 respectively. Based on the reported findings, it can be suggested that C. latifolia plants have properties that could be used to make sunscreen products and protect the skin against erythema, pigmentation, and harmful UV rays.98\n\n\n8. Conclusion\n\nThis review provides information on the distribution, species description, traditional uses, nutritional value, phytochemistry, chemical composition, and pharmacological activities of Curculigo latifolia. Previous studies have described the plant as a good source of phytochemical compounds, such as alkaloids, tannins, saponins, flavonoids, glycosides, steroids, terpenoids, and norlignans. Several common compounds, such as curculigoside, crassifoside I, nyasicoside, and curculigine, were also detected, which could play a significant role in the biological activities of C. latifolia. Furthermore, the plant has been investigated for several pharmacological activities such as antioxidant, antimicrobial, anti–diabetic, cholesterol, anti–elastase, ultraviolet protection, spermatogenic and cytotoxic activities.\n\nConsidering the promising biological studies outlined, C. latifolia could mitigate some diseases and therefore have potential medicinal value that could be used as dietary food supplements. However, the underlying potential of C. latifolia is not yet fully understood, and merits further research. Further investigations could include studies of other bioactivities, such as anti–cancer, anti–viral, wound healing, and anti–inflammatory activities. Isolation and characterization of the phytochemicals in the different parts of C. latifolia with varying extraction solvents should also be undertaken to identify bioactive compounds and further understand their mechanisms of action and biological activities. Moreover, human clinical trials could provide more information on the plant as a functional food for improving human health.\n\n\nDeclaration\n\nNot applicable – review paper.\n\n\nConsent for publication\n\nNot applicable - does not contain data from any individual person.\n\n\nAuthors’ contributions\n\nAT, HY, NA, MA, and FJ have made substantial contributions to the conception and design of the manuscript; have drafted the work and substantively revised it; have approved the submitted version (and any substantially modified version that involves the author’s contribution to the study); have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even those in which the author was not personally involved, are appropriately investigated and resolved, and the resolution documented in the literature.\n\nAll authors read and approved the final manuscript.", "appendix": "Data availability statement\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe authors would like to thank Collaborative Professor Hjh Normah Jamil, Osaka University ASEAN Campus Brunei Darussalam, Professor Quamrul Hasan, and Professor Yoshinori Sumimura from the Center for Global Initiatives, Osaka University, Japan for facilitating the Osaka University ASEAN Campus Universiti Brunei Darussalam collaboration.\n\n\nReferences\n\nNie Y, Dong X, He Y, et al.: Medicinal Plants of Genus Curculigo: Traditional Uses and a Phytochemical and Ethnopharmacological Review. J. Ethnopharmacol. June 3, 2013; 147: 547–563. PubMed Abstract \| Publisher Full Text\n\nWang Y, Li J, Li N: Phytochemistry and Pharmacological Activity of Plants of Genus Curculigo: An Updated Review since 2013. Molecules. June 1, 2021; 26. MDPI AG. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFern K: (accessed 2023-08-14). Reference Source\n\nBriliani M, Perdana C, Yuan Perwirasari I, et al.: Potency of Curculigo Capitulata and Curculigo Latifolia Fruit Based on Nutrient Content (a Case for Preservation Need in Kebonharjo). Biotropic J. Trop. Biol. 2018; 2(2): 67–72. Publisher Full Text\n\nNwokonkwo DC: Antibacterial Susceptibility of the Constituents of Ethanol Crude Extract and the Neutral Metabolite of the Root of Curculigo Pilosa Hypoxidaceae. Int. J. Chem. 2014; 6(4): 19–23. Publisher Full Text\n\nLiu M, Li Y, Yang ST: Curculigoside Improves Osteogenesis of Human Amniotic Fluid-Derived Stem Cells. Stem Cells Dev. 2014; 23(2): 146–154. PubMed Abstract \| Publisher Full Text\n\nSingla K, Singh R: Nephroprotective Effect of Curculigo Orchiodies in Streptozotocin-Nicotinamide Induced Diabetic Nephropathy in Wistar Rats. J. Ayurveda Integr. Med. 2020; 11(4): 399–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang K-J, Ning L: Norlignan Derivatives from Curculigo Crassifolia and Their DPPH Radical Scavenging Activity. Arch. Pharm. Res. 2008; 31(10): 1313–1316. PubMed Abstract \| Publisher Full Text\n\nXie Y, Chen J, Xiao A, et al.: Antibacterial Activity of Polyphenols: Structure-Activity Relationship and Influence of Hyperglycemic Condition. Molecules. 2017; 22(11): 1913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZabidi NA, Ishak NA, Hamid M, et al.: Inhibitory Evaluation of Curculigo Latifolia on α-Glucosidase, DPP (IV) and in vitro Studies in Antidiabetic with Molecular Docking Relevance to Type 2 Diabetes Mellitus. J. Enzyme Inhib. Med. Chem. 2021; 36(1): 109–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu F-B, Wang J-Y, Zhang Y-L, et al.: Curculigoside Regulates Proliferation, Differentiation, and pro-Inflammatory Cytokines Levels in Dexamethasone-Induced Rat Calvarial Osteoblasts. Int. J. Clin. Exp. Med. 2015; 8(8): 12337–12346. PubMed Abstract\n\nWorld Flora Online: Curculigo latifolia Dryand. ex W.T.Aiton. (accessed 2023-08-14). Reference Source\n\nLim TK: Edible Medicinal and Non-Medicinal Plants. Springer; 2012; vol. 4. . Publisher Full Text\n\nPlants of the World Online: Curculigo latifolia Dryand. ex W.T.Aiton. (accessed 2023-08-14). Reference Source\n\nFarzinebrahimi R, Mat Taha R, Rashid KA, et al.: Preliminary Screening of Antioxidant and Antibacterial Activities and Establishment of an Efficient Callus Induction in Curculigo Latifolia Dryand (Lemba). Evid. Based Complement. Alternat. Med. 2016; 2016: 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarzinebrahimi R, Taha RM, Rashid KA: Effect of Light Intensity and Soil Media on Establishment and Growth of Curculigo Latifolia Dryand. J. Appl. Hortic. 2013; 15(3): 224–226. Publisher Full Text\n\nFern K: Tropical Plants Database: Molineria latifolia. (accessed 2023-08-14). Reference Source\n\nAkkarasiritharattana K, Chamutpong S: Phytochemistry and Antioxidant Activity of Molineria Latifolia Herb. Extracts. Appl. Mech. Mater. 2019; 891: 21–29. Publisher Full Text\n\nLim SH, Ibrahim D: Studies on Antibiotic Compounds of Methanol Extract of Curculigo Latifolia Dryand. Proceedings of the 2nd Annual International Conference Syiah Kuala University 2012 & The 8th IMT-GT Uninet Biosciences Conference. 2012; Vol. 2: pp. 383–385.\n\nOoi DJ, Chan KW, Sarega N, et al.: Bioprospecting the Curculigoside-Cinnamic Acid-Rich Fraction from Molineria Latifolia Rhizome as a Potential Antioxidant Therapeutic Agent. Molecules. 2016; 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulip J: An Ethnobotanical Survey of Medicinal and Other Useful Plants of Muruts in Sabah, Malaysia. Telopea (Syd). 2003; 10(1): 81–98. Publisher Full Text\n\nMohiddin MYBH, Chin W, Holdsworth D: Traditional Medicinal Plants of Brunei Darussalam Part II Sengkurong. Int. J. Pharmacogn. 2008; 29(4): 252–258. Publisher Full Text\n\nMaliwong J, Chimnoi N, Ruchirawat S, et al.: Phenolic Glycosides from the Roots of Molineria Latifolia. Phytochem. Lett. 2021; 46: 90–94. Publisher Full Text\n\nIshak NA, Zabidi NA: Hypoglycemic Activity of Curculigo Latifolia on Insulin and Glucose Uptake Activities in in Vitro. J. Eng. Appl. Sci. 2019.\n\nGusmalawati D, Mayasari E: Karakteristik Fisikokimiawi Sari Buah Tapus (Curculigo Latifolia Dryand) Dengan Metode Ekstraksi Osmosis. Jurnal Ilmiah Teknosains. 2017; 3(2). Publisher Full Text\n\nDwivedi RS: Source of Curculin: Curculigo Latifolia (Dryand Ex W.T. Aiton). Alternative Sweet and Supersweet Principles: Natural Sweeteners and Plants. 2022; p. 571. Publisher Full Text\n\nKoizumi T, Terada T, Nakajima KI, et al.: Identification of Key Neoculin Residues Responsible for the Binding and Activation of the Sweet Taste Receptor. Sci. Rep. 2015; 5: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurimoto E, Suzuki M, Amemiya E, et al.: Curculin Exhibits Sweet-Tasting and Taste-Modifying Activities through Its Distinct Molecular Surfaces. J. Biol. Chem. 2007; 282(46): 33252–33256. PubMed Abstract \| Publisher Full Text\n\nShimizu-Ibuka A, Morita Y, Terada T, et al.: Crystal Structure of Neoculin: Insights into Its Sweetness and Taste-Modifying Activity. J. Mol. Biol. 2006; 359(1): 148–158. PubMed Abstract \| Publisher Full Text\n\nShaari N: Lemba (Curculigo Latifolia) Leaf as a New Materials for Textiles.2005.\n\nRaden I, Nugroho CC, Syahrani.: Identification and Characterization of Morphological Diversity of Lemba (Curculigo Latifolia) in East Kalimantan, Indonesia. Biodiversitas. 2017; 18(4): 1367–1376. Publisher Full Text\n\nAndansari D, Pristanti H, Keliwar S, et al.: The Development of Ulap Doyo Handheld Handbag for Application in Pokant Takaq Small and Medium Enterprise, Kalimantan Timur to Support Tourism Sector. Proceedings of the International Conference on Applied Science and Technology on Social Science 2021 (iCAST-SS 2021). 2022; 647. Publisher Full Text\n\nNational Parks Flora and Fauna: Molineria latifolia (Dryand. ex W.T.Aiton) Herb. ex Kurz. (accessed 2023-08-14). Reference Source\n\nBabaei N, Psyquay Abdullah NA, Saleh G, et al.: An Efficient in vitro Plantlet Regeneration from Shoot Tip Cultures of Curculigo Latifolia, a Medicinal Plant. Sci. World J. 2014; 2014: 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaryanto H, Sutandi A, Kusumawati E, et al.: Potential of Therapeutic Curculigo Latifolia Extracts on Alloxan-Induced Diabetes in a Male Mus Muscullus. Biosaintifika. 2023; 15(3): 370–377. Publisher Full Text\n\nNur S, Setiawan H, Hanafi M, et al.: Pharmacognostical and Phytochemical Studies and Biological Activity of Curculigo Latifolia Plant Organs for Natural Skin-Whitening Compound Candidate. Sci. World J. 2023; 2023: 1–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUmar AH, Ratnadewi D, Rafi M, et al.: Phenolics Profile and Antioxidant Activities of in vitro Propagules and Field-Raised Plant Organs of Curculigo Latifolia. J. Appl. Pharm. Sci. 2023; 13(4): 168–185. Publisher Full Text\n\nUmar AH, Ratnadewi D, Rafi M, et al.: Callus of Curculigo Latifolia Dryand. Ex W.T. Aiton: Initiation, Regeneration, Secretory Structure and Histochemistry. IOP Conf. Ser. Earth Environ. Sci. 2021; 948(1): 012051. Publisher Full Text\n\nUmar AH, Ratnadewi D, Rafi M, et al.: Metabolite Profiling, Distribution of Secretory Structures, and Histochemistry in Curculigo Orchioides Gaertn. And Curculigo Latifolia Dryand. Ex w.t.Aiton. Turk J Botany. 2021; 45(5): 421–439. Publisher Full Text\n\nGharibzahedi SMT, Jafari SM: The Importance of Minerals in Human Nutrition: Bioavailability, Food Fortification, Processing Effects and Nanoencapsulation. Trends Food Sci. Technol. 2017; 62: 119–132. Publisher Full Text\n\nSoni N, Mehta S, Satpathy G, et al.: Estimation of Nutritional, Phytochemical, Antioxidant and Antibacterial Activity of Dried Fig (Ficus Carica). J Pharmacogn Phytochem. 2014; 3(2): 158–165.\n\nOyeyinka BO, Afolayan AJ: Comparative Evaluation of the Nutritive, Mineral, and Antinutritive Composition of Musa Sinensis L. (Banana) and Musa Paradisiaca L. (Plantain) Fruit Compartments. Plants. 2019; 8(12): 598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVormann J: Magnesium: Nutrition and Homoeostasis. AIMS Public Health. 2016; 3(2): 329–340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahesh DS, Vidhathri BS, Vidyashree DN, et al.: Biochemical Composition and Pharmacological Properties of Mulberry (Morus Spp.) - A Review. Int. J. Curr. Microbiol. App. Sci. 2017; 6(7): 2207–2217. Publisher Full Text\n\nPravina P, Sayaji D, Avinash M: Calcium and Its Role in Human Body. Int. J. Res. Pharmaceut. Biomed. Sci. 2013; 4(2).\n\nGodswill AG, Somtochukwu IV, Ikechukwu AO, et al.: Health Benefits of Micronutrients (Vitamins and Minerals) and Their Associated Deficiency Diseases: A Systematic Review. Int. J. Food Sci. 2020; 3(1): 1–32. Publisher Full Text\n\nCormick G, Belizán JM: Calcium Intake and Health. Nutrients. 2019; 11(7): 1606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbaspour N, Hurrell R, Kelishadi R: Review on Iron and Its Importance for Human Health. J. Res. Med. Sci. 2014; 19(2): 164–174. PubMed Abstract\n\nAlajil O, Sagar VR, Kaur C, et al.: Nutritional and Phytochemical Traits of Apricots (Prunus Armeniaca L.) for Application in Nutraceutical and Health Industry. Foods. 2021; 10(6): 1344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMariyam P, Mary V: Nutritional Analysis (Macronutrients, Potassium and Iron Content) of Four Palm Date Varieties (Phoenix Dactylifera L.) and Study of Consumption Pattern among Muslim and Maharashtrian Community. J. Food Process. Beverages. 2014; 3(1).\n\nYakasai J, Yakasai BA, Aghanwa C: Effects of Freezing Period on the PH, Crude Protein and Some Essential Minerals in Catfish (Clarias Gariepinus) and Tilapia (Oreochromis Niloticus) Cultivated in Kaduna Metropolis, Kaduna State, Nigeria A. Int. J. Sci. Res. 2018; 7(5).\n\nOliveira F, Rocha S, Fernandes R: Iron Metabolism: From Health to Disease. J. Clin. Lab. Anal. 2014; 28(3): 210–218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli AAH: Overview of the Vital Roles of Macro Minerals in the Human Body. J. Trace Elem. Miner. 2023; 4: 100076. Publisher Full Text\n\nLaville M, Nazare J-A: Diabetes, Insulin Resistance and Sugars. Obes. Rev. 2009; 10: 24–33. Publisher Full Text\n\nLumbangaol N: Penentuan Kadar Tanin Total Ekstrak Etanol Buah Marasi (Curculigo Latifolia) Dengan Metode Spektroscopy UV-Visible. J. Herb. Med. 2020; 3(2): 2621–2625.\n\nUmar AH, Ratnadewi D, Rafi M, et al.: Untargeted Metabolomics Analysis Using FTIR and UHPLC-Q-Orbitrap HRMS of Two CurculigoSpecies AndEvaluation of Their Antioxidant and α-GlucosidaseInhibitory Activities. Metabolites. 2021; 11(42). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamtiya M, Aluko RE, Dhewa T, et al.: Potential Health Benefits of Plant Food-Derived Bioactive Components: An Overview. Foods. 2021; 10(4): 839. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZabidi NA, Ishak NA, Hamid M, et al.: Subcritical Water Extraction of Antioxidants from Curculigo Latifolia Root.2019. Publisher Full Text\n\nTran N, Pham B, Le L: Bioactive Compounds in Anti-Diabetic Plants: From Herbal Medicine to Modern Drug Discovery. Biology (Basel). 2020; 9(9): 252. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlora SJ: Structural, Chemical and Biological Aspects of Antioxidants for Strategies against Metal and Metalloid Exposure. Oxidative Med. Cell. Longev. 2009; 2(4): 191–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaini R: Coenzyme Q10: The Essential Nutrient. J. Pharm. Bioallied Sci. 2011; 3(3): 466–467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNur S, Setiawan H, Hanafi M, et al.: Phytochemical Composition, Antioxidant, in vitro and in Silico Studies of Active Compounds of Curculigo Latifolia Extracts as Promising Elastase Inhibitor. Saudi J. Biol. Sci. 2023; 30(8): 103716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRazavi M, Zarrini G: Bioactivity of Aviprin and Aviprin-3″-O-Glucoside, Two Linear Furanocoumarins from Apiaceae. Bioorg. Khim. 2010; 36(3): 392–395. PubMed Abstract \| Publisher Full Text\n\nScozzafava A, Passaponti M, Supuran CT, et al.: Carbonic Anhydrase Inhibitors: Guaiacol and Catechol Derivatives Effectively Inhibit Certain Human Carbonic Anhydrase Isoenzymes (HCA I, II, IX and XII). J. Enzyme Inhib. Med. Chem. 2015; 30(4): 586–591. PubMed Abstract \| Publisher Full Text\n\nChu KT, Ng TB: Smilaxin, a Novel Protein with Immunostimulatory, Antiproliferative, and HIV-1-Reverse Transcriptase Inhibitory Activities from Fresh Smilax Glabra Rhizomes. Biochem. Biophys. Res. Commun. 2006; 340(1): 118–124. PubMed Abstract \| Publisher Full Text\n\nOch A, Podgórski R, Nowak R: Biological Activity of Berberine—A Summary Update. Toxins (Basel). 2020; 12(11): 713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen GL, Mutie FM, Xu YB, et al.: Antioxidant, Anti-Inflammatory Activities and Polyphenol Profile of Rhamnus Prinoides. Pharmaceuticals. 2020; 13(4): 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiopan V, Babula P, Shestivska V, et al.: Electrochemical and Spectrometric Study of Antioxidant Activity of Pomiferin, Isopomiferin, Osajin and Catalposide. J. Pharm. Biomed. Anal. 2008; 48(1): 127–133. PubMed Abstract \| Publisher Full Text\n\nDong J, Zhong T, Xu Z, et al.: Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model. Cancers (Basel). 2022; 14(19): 4710. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta M, Achari B, Pal BC: Glucosides from Curculigo Orchioides. Phytochemistry. 2005; 66(6): 659–663. Publisher Full Text\n\nLi N, Chen JJ, Zhao YX, et al.: Three New Norlignans from Curculigo Capitulata. J. Asian Nat. Prod. Res. 2005; 7(3): 189–195. PubMed Abstract \| Publisher Full Text\n\nWang KJ, Zhu CC, Di L, et al.: New Norlignan Derivatives from Curculigo Capitulata. Fitoterapia. 2010; 81(7): 869–872. PubMed Abstract \| Publisher Full Text\n\nLi N, Li SP, Wang KJ, et al.: Novel Norlignan Glucosides from Rhizomes of Curculigo Sinensis. Carbohydr. Res. 2012; 351: 64–67. PubMed Abstract \| Publisher Full Text\n\nMad Nasir N, Ezam Shah NS, Zainal NZ, et al.: Combination of Molecular Networking and Lc-Ms/Ms Profiling in Investigating the Interrelationships between the Antioxidant and Antimicrobial Properties of Curculigo Latifolia. Plants. 2021; 10(8). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGe JF, Gao WC, Cheng WM, et al.: Orcinol Glucoside Produces Antidepressant Effects by Blocking the Behavioural and Neuronal Deficits Caused by Chronic Stress. Eur. Neuropsychopharmacol. 2014; 24(1): 172–180. PubMed Abstract \| Publisher Full Text\n\nZhang Y, Ge JF, Wang FF, et al.: Crassifoside H Improve the Depressive-like Behavior of Rats under Chronic Unpredictable Mild Stress: Possible Involved Mechanisms. Brain Res. Bull. 2017; 135: 77–84. PubMed Abstract \| Publisher Full Text\n\nMuhit MA, Izumikawa M, Umehara K, et al.: Phenolic Constituents of the Bangladeshi Medicinal Plant Pothos Scandens and Their Anti-Estrogenic, Hyaluronidase Inhibition, and Histamine Release Inhibitory Activities. Phytochemistry. 2016; 121: 30–37. PubMed Abstract \| Publisher Full Text\n\nZeng J, Liu X, Li X, et al.: Daucosterol Inhibits the Proliferation, Migration, and Invasion of Hepatocellular Carcinoma Cells via Wnt/β-Catenin Signaling. Molecules. 2017; 22(6): 862. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXie Y, Peng Q, Ji Y, et al.: Isolation and Identification of Antibacterial Bioactive Compounds From Bacillus Megaterium L2. Front. Microbiol. 2021; 12: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNigam M: Phytomedicine: Scope and Current Highlights. Preparation of Phytopharmaceuticals for the Management of Disorders: The Development of Nutraceuticals and Traditional Medicine. 2021; pp. 39–54. Publisher Full Text\n\nAkinwumi B, Bordun K-A, Anderson H: Biological Activities of Stilbenoids. Int. J. Mol. Sci. 2018; 19(3): 792. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnand David AV, Arulmoli R, Parasuraman S: Overviews of Biological Importance of Quercetin: A Bioactive Flavonoid. Pharmacogn. Rev. 2016; 10(20): 84–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShuvalov O, Fedorova O, Tananykina E, et al.: An Arthropod Hormone, Ecdysterone, Inhibits the Growth of Breast Cancer Cells via Different Mechanisms. Front. Pharmacol. 2020; 11: 561537. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang JB, Yang CS, Li J, et al.: Dianthrone Derivatives from Polygonum Multiflorum Thunb: Anti-Diabetic Activity, Structure-Activity Relationships (SARs), and Mode of Action. Bioorg. Chem. 2023; 135: 106491. PubMed Abstract \| Publisher Full Text\n\nYang K, Bao T, Zeng J, et al.: Research Progress on Pyroptosis-Mediated Immune-Inflammatory Response in Ischemic Stroke and the Role of Natural Plant Components as Regulator of Pyroptosis: A Review. Biomed. Pharmacother. 2023; 157: 113999. PubMed Abstract \| Publisher Full Text\n\nPalmeri A, Mammana L, Tropea MR, et al.: Salidroside, a Bioactive Compound of Rhodiola Rosea, Ameliorates Memory and Emotional Behavior in Adult Mice. J. Alzheimers Dis. 2016; 52(1): 65–75. PubMed Abstract \| Publisher Full Text\n\nWeng MS, Ho CT, Ho YS, et al.: Theanaphthoquinone Inhibits Fatty Acid Synthase Expression in EGF-Stimulated Human Breast Cancer Cells via the Regulation of EGFR/ErbB-2 Signaling. Toxicol. Appl. Pharmacol. 2007; 218(2): 107–118. PubMed Abstract \| Publisher Full Text\n\nShahidi F, Zhong Y: Measurement of Antioxidant Activity. J. Funct. Foods. 2015; 18: 757–781. Publisher Full Text\n\nTungmunnithum D, Thongboonyou A, Pholboon A, et al.: Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines. 2018; 5(3): 93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharifi-Rad M, Anil Kumar NV, Zucca P, et al.: Lifestyle, Oxidative Stress, and Antioxidants: Back and Forth in the Pathophysiology of Chronic Diseases. Front. Physiol. 2020; 11: 552535. Publisher Full Text\n\nLim S-H, Ibrahim D: Assessment of Anticandidal Activity and Cytotoxicity of Root Extract from Curculigo Latifolia on Pathogenic Candida Albicans. J. Med. Sci. 2013; 13(3): 193–200. Publisher Full Text\n\nIshak NA, Ismail M, Hamid M, et al.: Antidiabetic and Hypolipidemic Activities of Curculigo Latifolia Fruit:Root Extract in High Fat Fed Diet and Low Dose STZ Induced Diabetic Rats. Evid. Based Complement. Alternat. Med. 2013; 2013: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOoi DJ, Azmi NH, Imam MU, et al.: Curculigoside and Polyphenol-Rich Ethyl Acetate Fraction of Molineria Latifolia Rhizome Improved Glucose Uptake via Potential MTOR/AKT Activated GLUT4 Translocation. J. Food Drug Anal. 2018; 26(4): 1253–1264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOoi DJ, Adamu HA, Imam MU, et al.: Polyphenol-Rich Ethyl Acetate Fraction Isolated from Molineria Latifolia Ameliorates Insulin Resistance in Experimental Diabetic Rats via IRS1/AKT Activation. Biomed. Pharmacother. 2018; 98: 125–133. PubMed Abstract \| Publisher Full Text\n\nJaafar FM, Zainal FS, Ahmat N, et al.: The Effects of Curculigo Latifolia Dryand Ethanolic Extracts on Sperm Quality of Mice Mus Musculus. ESTEEM Academic Journal. 2017; 13: 75–82.\n\nRiss T, Niles A, Moravec R, et al.: Cytotoxicity Assays: in vitro Methods to Measure Dead Cells. Assay Guidance Manual. 2019.\n\nNur S, Hanafi M, Setiawan H, et al.: In Silico Evaluation of the Dermal Antiaging Activity of Molineria Latifolia (Dryand. Ex W.T. Aiton) Herb. Ex Kurz Compounds. J. Pharm. Pharmacogn. Res. 2023; 11(2): 325–345. Publisher Full Text\n\nNur S, Hanafi M, Setiawan H, et al.: In Vitro Ultra Violet (UV) Protection of Curculigo Latifolia Extract as a Sunscreen Candidate. IOP Conf. Ser. Earth Environ. Sci. 2022; 1116(1): 012009. Publisher Full Text" }	[ { "id": "281841", "date": "24 Jun 2024", "name": "Azlini Ismail", "expertise": [ "Reviewer Expertise Antihypertensive", "antioxidant", "anti-inflammatory", "pharmacognosy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this review article summarizes the phytochemicals, nutritional, and biological properties of Curculigo latifolia and highlights the gap of studies required for future research.\nThere are a few amendments required for further improvement:\nIntroduction – Please correct the typographical error for some words (i.e. ome) Figure 1 (I) should be standardized using small letter (i). The labels (c) and (d) for roots and petioles/stem are switched. Please double-check. Please double-check grammar for the following: regulation blood pressure. To re-confirm the following sentence: …..the plant was shown to exhibit bioactivities such as antioxidant, antimicrobial, anti–diabetic, sperm quality, cytotoxicity, anti–aging and ultraviolet protection activities. Sperm quality is not a type of bioactivity, kindly choose other suitable words. The findings using the brine shrimp test and MTT assay indicate that the plant was not cytotoxic, thus it is better to omit the cytotoxic effect in this sentence and wherever applicable in this review, unless some findings suggest so. Please revise the following sentence as the diverse range of bioactivities is attributed to the presence of different types of bioactive compounds that the plant has, not because of the high content of any chemical compounds: The diverse range of bioactivities of plants may be attributed to their high content of chemical compounds. Please correct the sentence (IC50 11.49 ± 0.071 μg/mL). Please double-check the value in the following sentence: Furthermore, a study by Mad Nasir et al.74 revealed that the methanolic leaf extract had promising antibacterial properties against Staphylococcus aureus (gram-positive bacteria) and Salmonella choleraesuis (gram-negative bacteria), with inhibition zones of ±15.33 mm and ±8 mm, respectively. Please revise the typographical error in the following sentence: The study found that The hexane leaf extract demonstrated the highest intensity in absorbing UVA and UVB radiation, with absorption values of 1.192 and 1.804, respectively.\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": "12043", "date": "23 Jul 2024", "name": "Amanina Taufik", "role": "Author Response", "response": "Dear Reviewer, We appreciate your thoughtful and thorough review of our manuscript. We have carefully considered your feedback and made the following revisions (please see below): Comment 1: Introduction – Please correct the typographical error for some words (i.e. ome) Response 1: Thank you for noting the error. The typo has been corrected: “Some species in this genus are known for their medicinal properties and have often been used in traditional therapeutic remedies.” Comment 2: Figure 1 (I) should be standardized using small letter (i). The labels (c) and (d) for roots and petioles/stem are switched. Please double-check. Response 2: Thank you for your comment. (I) has been changed to small letter (i) and the labels for (c ) and (d) have been switched. Comment 3: Please double-check grammar for the following: regulation blood pressure. Response 3: Thank you for pointing out the error. The sentence has been corrected to: “regulation of blood pressure.” Comment 4: To re-confirm the following sentence: …..the plant was shown to exhibit bioactivities such as antioxidant, antimicrobial, anti–diabetic, sperm quality, cytotoxicity, anti–aging and ultraviolet protection activities. Sperm quality is not a type of bioactivity, kindly choose other suitable words. The findings using the brine shrimp test and MTT assay indicate that the plant was not cytotoxic, thus it is better to omit the cytotoxic effect in this sentence and wherever applicable in this review, unless some findings suggest so. Response 4: Thank you for your comment. We agree that sperm quality is not a type of bioactivity and therefore, this sentence has been rewritten to, “C. latifolia has been involved in several in vitro and in vivo investigations, where the plant was shown to exhibit bioactivities such as antioxidant, antimicrobial, anti–diabetic, anti–aging, ultraviolet protection activities and reported to improve sperm quality (summarised in Table 4).” Additionally, we have replaced the term “sperm quality” in Table 4 which listed the reported biological activities to “spermatogenic activity.” We have also removed cytotoxic effect from the sentence. Comment 5: Please revise the following sentence as the diverse range of bioactivities is attributed to the presence of different types of bioactive compounds that the plant has, not because of the high content of any chemical compounds: The diverse range of bioactivities of plants may be attributed to their high content of chemical compounds. Response 5: Thank you for your comment. The sentence has been revised to: “The diverse range of bioactivities of plants may be attributed to the different types of bioactive compounds.” Comment 6: Please correct the sentence (IC50 11.49 ± 0.071 μg/mL). Response 6: Thank you for your comment. The sentence has been revised to: “The report showed that the rhizome extract produced a high DPPH radical scavenging activity having an IC 50 value of 18.10 ± 0.91 μg/mL, which is comparable to that of standard ascorbic acid, which has an IC 50 value of 11.49 ± 0.071 μg/mL).” Comment 7: Please double-check the value in the following sentence: Furthermore, a study by Mad Nasir et al.74 revealed that the methanolic leaf extract had promising antibacterial properties against Staphylococcus aureus (gram-positive bacteria) and Salmonella choleraesuis (gram-negative bacteria), with inhibition zones of ±15.33 mm and ±8 mm, respectively. Response 7: Thank you for your comment. The values were changed and revised to: “Furthermore, a study by Mad Nasir et al. 74 revealed that the methanolic leaf extract had promising antibacterial properties against Staphylococcus aureus (gram-positive bacteria) and Salmonella choleraesuis (gram-negative bacteria), with mean (n=3) inhibition zones of 15.33 mm and 8 mm, respectively.” Comment 8: Please revise the typographical error in the following sentence: The study found that The hexane leaf extract demonstrated the highest intensity in absorbing UVA and UVB radiation, with absorption values of 1.192 and 1.804, respectively. Response 8: Thank you for pointing out the error. The typo has been corrected: “The study found that the hexane leaf extract demonstrated the highest intensity in absorbing UVA and UVB radiation, with absorption values of 1.192 and 1.804, respectively.”" } ] } ]	1	https://f1000research.com/articles/13-495
https://f1000research.com/articles/13-502/v1	17 May 24	{ "type": "Study Protocol", "title": "An observational cross-sectional study of the role of magnetic resonance imaging in the evaluation of T2-weighted hyperintensities in the spinal cord in tertiary care hospital in central India", "authors": [ "Rishabh Dhabalia", "Shivali Kashikar", "Shivali Kashikar" ], "abstract": "Background T2-weighted hyperintensities in the spinal cord are a complex and diagnostically challenging entity that can present with diverse clinical features. This study protocol outlines a comprehensive investigation to understand the causes, clinical and imaging characteristics, and correlation with pathological findings of T2-weighted hyperintensities in the spinal cord. By establishing a systematic assessment approach, this study seeks to provide valuable insights into these abnormalities’ diagnostic and prognostic implications.\n\nMethods The study will be conducted as a prospective observational design. Patients with clinically diagnosed or suspected spinal cord injury presenting with intramedullary T2-weighted hyperintensity and referred for MRI evaluation will be included. Data collection will encompass patient demographics, clinical features, and extensive imaging parameters. Pathological data, when available, will be correlated with imaging findings. Various statistical methods will be employed to analyse the data, including frequency analysis, comparative tests, logistic regression, and survival analysis.\n\nExpected Results The study anticipates elucidating the spectrum of etiologies underlying T2-weighted hyperintensities in the spinal cord and their clinical and imaging profiles. The systematic approach will offer a structured diagnostic method, while correlations with pathological data will provide an enhanced understanding of these conditions. The results are expected to provide clinicians with valuable insights into diagnosing, treating, and prognosticating patients with spinal cord hyperintensities.", "keywords": [ "Spinal Cord", "T2-Weighted Hyperintensities", "MRI Evaluation", "Clinical Features", "Pathological Correlation", "Systematic Assessment" ], "content": "Introduction\n\nT2-weighted hyperintensities within the spinal cord represent a diverse and challenging diagnostic entity, the understanding of which is critical for improving patient care and outcomes.1 These abnormalities manifest with various clinical symptoms, making their etiological diagnosis a complex and vital task. Magnetic resonance imaging (MRI) is the cornerstone for evaluating spinal cord pathologies, offering a window into the intricate details of the spinal cord’s internal structure and any potential anomalies.2 In this context, the current study protocol is designed to investigate T2-weighted hyperintensities in the spinal cord comprehensively. It focuses on identifying their causes, analysing clinical and imaging features, developing a systematic diagnostic approach, and correlating with pathological data when available.\n\nSpinal cord pathologies can lead to many clinical presentations, from sensory and motor deficits to more complex neurological signs, impacting affected individuals’ quality of life and prognosis. The ability to accurately identify and diagnose the underlying causes of T2-weighted hyperintensities in the spinal cord is crucial for timely intervention and treatment planning.3 Moreover, understanding the relationships between clinical features, imaging characteristics, and pathological findings can provide a holistic perspective on these conditions, facilitating more precise diagnosis and improved patient care.4\n\nThe study aims to provide valuable insights into the spectrum of etiologies of T2-weighted hyperintensities within the spinal cord and their correlation with clinical and imaging features. Developing a systematic, algorithmic approach for their assessment will enhance diagnostic accuracy and offer a structured framework for clinicians. Furthermore, when pathological data is available, this study will bridge the gap between imaging and histopathological findings, potentially shedding light on the mechanistic aspects of these abnormalities.\n\nUltimately, the findings from this investigation are expected to contribute to a better understanding of T2-weighted hyperintensities in the spinal cord, facilitating early diagnosis, treatment, and improved clinical-neurological outcomes for affected patients. By addressing the complex diagnostic challenges associated with these abnormalities, this study protocol seeks to advance our knowledge and clinical management of spinal cord pathologies, further underscoring the significance of MRI evaluation in this context.\n\nThis study aims to investigate the prevalence and impact of T2-weighted hyperintensities in the spinal cord, determine their etiological factors, establish a systematic assessment approach, and correlate clinical and imaging features with neurological outcomes.\n\n\n\n1. To determine the causes of T2-weighted hyperintensities in the spinal cord.\n\n2. To analyse the clinical and imaging characteristics associated with different etiologies of T2-weighted hyperintensities in the spinal cord.\n\n3. To develop a systematic, algorithmic approach for evaluating intramedullary T2-weighted hyperintensities.\n\n4. To assess the morphological aspects of spinal cord hyperintensities and correlate findings with pathological data where available.\n\n5. To establish a diagnostic approach to intrinsic spinal cord T2 signal abnormalities and their relationship to clinical-neurological outcomes.\n\n\nMethods\n\nProspective Observational Study. Patients with clinically diagnosed or suspected spinal cord injury, presenting with intramedullary T2-weighted hyperintensities, will undergo an MRI evaluation of the spinal cord. Data will be collected in setup 2023-2024 period, and statistical analyses will be performed to achieve the study objectives.\n\nPatients with clinically diagnosed or suspected spinal cord injury, irrespective of age or gender, were referred to the Department of Radiodiagnosis, AVBRH, Sawangi, and presenting with intramedullary T2-weighted hyperintensity in the spinal cord.\n\nDepartment of Radiodiagnosis, AVBRH (Datta Meghe Institute of Higher Education & Research), Sawangi (Wardha).\n\n\n\n1. Clinically diagnosed or suspected patients with spinal cord injury are referred to the Department of Radiodiagnosis for MRI evaluation of the spinal cord.\n\n2. Patients with intramedullary T2-weighted hyperintensity.\n\n3. Patients of all age groups and both genders.\n\n4. Patients willing to undergo investigations, participate in the study, and agree to followup if required.\n\n\n\n1. Patients with cardiac pacemakers, metallic implant insertion, or metallic foreign bodies in situ.\n\n2. Patients with a history of claustrophobia.\n\n3. Patients who refuse to provide informed consent.\n\n4. Non-cooperative patients.\n\n\n\n1. Selection bias: There may be a potential bias in selecting study participants. If patients are not randomly selected and instead are chosen based on the severity of their condition or other non-random criteria, this can introduce selection bias. For example, patients with more severe symptoms may be more likely to undergo MRI evaluation, potentially leading to overrepresenting specific etiologies of T2-weighted hyperintensities.\n\nOvercoming selection bias:\n\n• Random sampling: To reduce selection bias, employ random sampling methods to ensure that all eligible patients have an equal chance of being included in the study.\n\n• Clear inclusion criteria: Clearly define the inclusion criteria to ensure consistency in patient selection.\n\n2. Observer bias: This bias can occur during the interpretation of MRI scans. If the radiologists or clinicians interpreting the MRI scans are aware of the patient’s clinical history or if they have preconceived notions about the potential cause of T2-weighted hyperintensities, this may influence their assessments.\n\nOvercoming observer bias:\n\n• Blinding: Implement blinding procedures where the radiologists or clinicians interpreting the MRI scans are unaware of the patient’s clinical history and etiological factors.\n\n• Inter-rater agreement: Ensure that multiple radiologists or clinicians independently assess the MRI scans and measure inter-rater agreement to minimise bias.\n\n3. Information bias: Information bias may arise if there are inaccuracies in data collection, documentation, or patient reporting. Only accurate or complete data can lead to errors in the study’s findings.\n\nOvercoming information bias:\n\n• Standardized data collection: Use standardised data collection forms and procedures to ensure consistent and accurate data collection.\n\n• Patient education: Educate patients about providing accurate and complete information, including medical history and symptoms.\n\n\n\nThe primary outcome of the study:\n\n1. Identification of causes: Determining the various underlying causes of T2-weighted hyperintensities in the spinal cord.\n\nSecondary outcomes:\n\n2. Clinical and imaging features: Analyzing the clinical and imaging characteristics associated with different causes of hyperintensities.\n\n3. Diagnostic approach: Establishing a systematic, algorithmic approach for assessing intramedullary T2-weighted hyperintensities.\n\n4. Pathological correlation: Correlating imaging findings with pathological data, whenever possible, to enhance diagnostic accuracy.\n\n5. Neurological outcomes: Assessing the correlation between spinal cord hyperintensities and clinical-neurological outcomes, potentially aiding prognosis and treatment decisions.\n\n\n\n1. Identification of eligible patients: The enrollment process will begin with identifying patients who meet the inclusion criteria for the study. This includes individuals with clinically diagnosed or suspected spinal cord injury referred to the Department of Radiodiagnosis, AVBRH, Sawangi, and present with intramedullary T2-weighted hyperintensity in the spinal cord.\n\n2. Patient education and informed consent: Patients identified as potentially eligible for the study will be approached by the study investigators or healthcare providers responsible for their care. They will be informed about the study’s purpose, procedures, potential risks, and benefits. Patients will be provided with both verbal and written information about the study. They will have the opportunity to ask questions and seek clarification.\n\n3. Obtaining informed consent: Patients willing to participate in the study will be asked to provide written informed consent. The informed consent process will be conducted in a private and confidential setting to ensure that patients fully understand the study and voluntarily agree to participate. Those who decline to participate or do not meet the inclusion criteria will be excluded.\n\n4. Proforma completion: The healthcare providers or study investigators will fill out a proforma after obtaining informed consent. This proforma will collect relevant patient information, including demographic data, clinical history, and other essential details necessary for the study.\n\n5. MRI evaluation: After enrollment, eligible patients will undergo an MRI evaluation of the spinal cord. The imaging will be performed per the standard protocols and procedures established by the Department of Radiodiagnosis.\n\n6. Regular monitoring: Throughout the enrollment process, there will be ongoing monitoring to ensure that all eligible patients are approached, informed, and allowed to participate. Any challenges or issues related to enrollment will be documented and addressed promptly.\n\n7. Data management: All collected data, including patient information and MRI findings, will be managed securely and confidentially, with access limited to authorised study personnel only.\n\nThe data collection process for this study is a critical component that ensures the systematic and accurate gathering of information about the objectives. It involves several stages designed to maintain the highest data quality standards and ethical considerations.\n\n1. Patient information and informed consent: Upon enrollment, patients who have provided informed consent will have their demographic information recorded. This includes age, gender, medical history, and any pertinent clinical details related to their spinal cord injury. The informed consent process is confidential to ensure that participants understand the study’s purpose and voluntarily agree to participate.\n\n2. Imaging data acquisition: The core of this study relies on magnetic resonance imaging (MRI) to assess T2-weighted hyperintensities in the spinal cord. Imaging data will be collected using state-of-the-art MRI equipment following established protocols. This imaging process will generate detailed images of the spinal cord and surrounding structures.\n\n3. Clinical and imaging features: Clinical features will be documented, including presenting symptoms, duration of symptoms, and any neurological deficits. Imaging features, such as the location, size, and characteristics of T2-weighted hyperintensities, will be recorded by experienced radiologists. The collected clinical and imaging data will provide valuable insights into the presentation and characteristics of spinal cord hyperintensities.\n\n4. Pathological data and correlation: Whenever possible, pathological data will be collected through procedures like biopsies or surgeries. This data will be correlated with the imaging findings, allowing for a comprehensive understanding of the underlying causes of T2-weighted hyperintensities.\n\n5. Proforma completion: Healthcare providers or study investigators will fill out a standardised proforma. This proforma will collect detailed information, ensuring the systematic and uniform documentation of all relevant patient data. It acts as a bridge between clinical and imaging information, aiding in the correlation process.\n\n6. Data entry and management: Trained personnel will enter All collected data into a secure and confidential database system. Strict data entry standards will be upheld to minimise errors and ensure data integrity. Access to the data will be restricted to authorised study personnel to maintain privacy and confidentiality.\n\n7. Quality control and validation: Data quality will be monitored through regular validation processes to identify any inconsistencies or inaccuracies. These will be promptly addressed to maintain the accuracy and reliability of the dataset.\n\n8. Ethical considerations: Ethical guidelines and patient confidentiality will be strictly adhered to throughout the data collection process. All patient data will be anonymised to protect privacy, and the study will comply with relevant data protection and patient confidentiality regulations.\n\nThe formula used for sample size calculation is as follows:\n\nIn this case, the sample size calculation was based on the following parameters:\n\n• Population size (N): 1,000,000 (estimated)\n\n• Hypothesized % frequency of the outcome (p): 17.5% ± 10\n\n• Confidence limits as % of 100 (absolute ± %) (d): 10%\n\n• Design effect (DEFF): 1\n\n• Confidence level (1-α/2): 99%\n\nGiven these parameters, the sample size was calculated to be 96. This sample size was determined to provide a 99% confidence level with a 10% margin of error while accounting for the estimated prevalence of the outcome in the population.\n\nFor this study on T2-weighted hyperintensities in the spinal cord, a comprehensive range of statistical methods will be employed to address the research objectives. The analysis will commence with descriptive statistics to provide an overview of the data’s central tendencies and distribution. Frequent analysis will be conducted to determine the prevalence of various causes of hyperintensities. Comparative analyses will involve chi-square tests, t-tests, and ANOVA to explore associations between clinical and imaging features and different causes of hyperintensities. Logistic regression will be applied to identify predictors and risk factors, while multinomial logistic regression will help assess factors associated with multiple categories of outcomes. Additionally, linear regression will be used to explore relationships between continuous outcomes and independent variables. Cox regression will address time-to-event data, such as clinical progression. Correlation analysis, survival analysis, and factor or cluster analysis will further assist in unveiling patterns and associations within the dataset. Comprehensive data visualisation will support the interpretation of findings. Subgroup and sensitivity analyses will also be conducted to explore variations across demographic or clinical subgroups and test the robustness of the results. Specialised statistical software will facilitate data analysis, and collaboration with a statistician will ensure these statistical techniques’ rigorous and appropriate application by using R Studio Version 4.3.1.\n\nThe Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted its approval to the study protocol (Reference number: DMIHER (DU)/IEC/2023/545. Date:03-02-2023). Before commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives.\n\nAfter the completion of the study, we will publish it in an indexed journal or conference.\n\nThe study has yet to start after the publication of the protocol; we will start recruitment in the study.\n\n\nDiscussion\n\nAs outlined in this study protocol, the evaluation of T2-weighted hyperintensities in the spinal cord holds significant clinical relevance. These hyperintensities present a diagnostic challenge due to their varied etiologies and clinical manifestations, necessitating a structured approach for assessment and correlation with pathological findings.\n\nThe systematic investigation of these intramedullary hyperintensities is aligned with the growing recognition of magnetic resonance imaging (MRI) as the gold standard for spinal cord evaluation. MRI offers high sensitivity, excellent contrast resolution, and the ability to visualise soft tissue structures in multiple planes, making it indispensable in assessing spinal cord disorders.5 Our study’s emphasis on T2-weighted hyperintensities extends its significance, as these abnormalities may harbour crucial clinical information.\n\nThe clinical and imaging features of T2-weighted hyperintensities in the spinal cord are central to understanding the impact on patients. The proposed study aims to provide insights into the characteristics associated with different etiologies of these hyperintensities. The ability to correlate imaging features with clinical symptoms can aid in early diagnosis and guide treatment decisions. Previous research has emphasised the diagnostic potential of MRI in identifying various spinal cord pathologies, including traumatic injuries, disc herniation, and inflammatory conditions.6,7 Our study seeks to contribute to this body of knowledge by examining the specific characteristics associated with T2-weighted hyperintensities.\n\nMoreover, developing a systematic, algorithmic approach for their assessment aligns with the call for standardised diagnostic protocols. A structured approach can improve diagnostic accuracy and consistency, ensuring patients receive appropriate care promptly. The utility of systematic approaches has been recognised in diagnosing spinal cord injuries and associated neurological deficits.8 In this context, our study aims to provide a framework that can be integrated into clinical practice, facilitating more precise and timely diagnoses.\n\nThe correlation of imaging findings with pathological data, whenever available, further enhances the study’s significance. This linkage between radiological and histopathological data may provide valuable insights into the underlying mechanisms of these hyperintensities.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nLee MJ, Aronberg R, Manganaro MS, et al.: Diagnostic Approach to Intrinsic Abnormality of Spinal Cord Signal Intensity. RadioGraphics. 2019; 39: 1824–1839. PubMed Abstract \| Publisher Full Text\n\nGbadamosi H, Mensah YB, Asiamah S: MRI features in the non-traumatic spinal cord injury patients presenting at the Korle Bu Teaching Hospital, Accra. Ghana Med. J. 2018; 52: 127–132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBennett J, Das MJ, Emmady PD: Spinal Cord Injuries. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023.\n\nBalogh EP, Miller BT, Ball JR: The Diagnostic Process Improving Diagnosis in Health Care. National Academies Press (US); 2015. Publisher Full Text\n\nMohajeri Moghaddam S, Bhatt AA: Location, length, and enhancement: systematic approach to differentiating intramedullary spinal cord lesions. Insights Imaging. 2018; 9: 511–526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTillema J-M, Pirko I: Neuroradiological evaluation of demyelinating disease. Ther. Adv. Neurol. Disord. 2013; 6: 249–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlawiter EC: Current and New Directions in MRI in Multiple Sclerosis. Contin. Lifelong Learn. Neurol. 2013; 19: 1058–1073. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalogh EP, Miller BT, Ball JR, et al.: Technology and Tools in the Diagnostic Process. In: Improving Diagnosis in Health Care. National Academies Press (US); 2015. Publisher Full Text" }	[ { "id": "284747", "date": "02 Jun 2024", "name": "Fabiano Reis", "expertise": [ "Reviewer Expertise Neuroradiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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.An observational cross-sectional study of the role of magnetic resonance imaging in the evaluation of the spinal cord in a tertiary care hospital in central India\n\nThe title is too long, I would suggest to remove some words. See above. 2.Hyperintensities on T2-weighted images in the spinal cord (use this form in the background). 3. In objectives, include:\n6. To establish a diagnostic approach considering the topography and extension of the spinal cord abnormalities. 4. include 3. Diagnostic approach: Establishing a systematic, algorithmic approach for assessing intramedullary T2-weighted hyperintensities, considering the topography and extension of the abnormalities. 5. USE THE WORD LESION This includes individuals with clinically diagnosed or suspected spinal cord lesion referred to the Department of Radiodiagnosis, 6.MRI evaluation: After enrollment, eligible patients will undergo an MRI evaluation of the spinal cord. The imaging will be performed per the standard protocols and procedures established by the Department of Radiodiagnosis. DETAIL THIS PROTOCOL 7. Imaging data acquisition: The core of this study relies on magnetic resonance imaging (MRI) to assess T2-weighted hyperintensities in the spinal cord. Imaging data will be collected using state-of-the-art MRI equipment following established protocols (DETAIL THE PROTOCOLS). 8. AFTER CONDITIONS, INCLUDE THIS INFORMATION AND THIS RESPECTIVE RECENT REFERENCE including traumatic injuries, disc herniation, inflammatory, and demyelinating conditions. The main imaging patterns of demyelinating myelopathies (multiple sclerosis, neuromyelitis Optica spectrum disorder, acute disseminated encephalomyelitis, and myelin oligodendrocyte glycoprotein antibody-related disorder) and inflammatory myelopathies (systemic lupus erythematosus-myelitis, sarcoidosis-myelitis, Sjogren-myelitis, and Bechet's-myelitis) have MRI features that can aid to obtain the right diagnosis. Tamanini JVG, Sabino JV, Cordeiro RA, Mizubuti V, Villarinho LL, Duarte JÁ, Pereira FV, Appenzeller S, Damasceno A, Reis F. The Role of MRI in Differentiating Demyelinating and Inflammatory (not Infectious) Myelopathies. Semin Ultrasound CT MR. 2023 Oct;44(5):469-488.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "11815", "date": "25 Jun 2024", "name": "Rishabh Dhabalia", "role": "Author Response", "response": "Thank you sir for your expert review. I have made the changes as per your advice." } ] } ]	1	https://f1000research.com/articles/13-502
https://f1000research.com/articles/12-427/v1	20 Apr 23	{ "type": "Research Article", "title": "A novel deep learning method for recognizing texts printed with multiple different printing methods", "authors": [ "Jarmo Koponen", "Keijo Haataja", "Pekka Toivanen", "Keijo Haataja", "Pekka Toivanen" ], "abstract": "Background: Text recognition of cardboard pharmaceutical packages with machine vision is a challenging task due to the different curvatures of packaging surfaces and different printing methods. Methods: In this research, a novel deep learning method based on regions with convolutional neural networks (R-CNN) for recognizing binarized expiration dates and batch codes printed using different printing methods is proposed. The novel method recognizes the characters in the images without the need to extract handcrafted features. In detail, this approach performs text recognition considering the whole image as an input extracting and learning salient character features straight from packaging surface images. Results: The expiration date and manufacturing batch codes of a real-life pharmaceutical packaging image set are recognized with 91.1% precision with a novel deep learning-based model, while Tesseract OCR text recognition performance with the same image set is 38.3%. The novel model outperformed Tesseract OCR also in tests evaluating recall, accuracy, and F-Measure performance. Furthermore, the novel model was evaluated in terms of multi-object recognition accuracy and the number of unrecognized characters, in order to achieve performance values comparable to existing multi-object recognition methods. Conclusions: The results of this study reveal that the novel deep learning method outperforms the well-established optical character recognition method in recognizing texts printed using different printing methods. The novel method presented in the study recognizes texts printed with different printing methods with high precision. The novel deep learning method is suitable for recognizing texts printed on curved surfaces with proper preprocessing. The problem investigated in the study differs from previous research in the field, focusing on the recognition of texts printed with different printing methods. The research thus fills a gap in text recognition that existed in the research of the field. Furthermore, the study presents new ideas that will be utilized in our future research.", "keywords": [ "Text recognition", "Machine Vision", "Deep Learning", "Regions with Convolutional Neural Networks", "R-CNN", "Character Recognition", "Printing Methods", "Expiration Date", "Batch Codes", "Handcrafted Features", "Image Recognition", "Multi-Object Recognition", "OCR", "Tesseract OCR", "Precision", "Recall", "Accuracy", "F-Measure", "Curved Surfaces", "Preprocessing" ], "content": "Introduction\n\nBy the end of 2023, the market value of the pharmaceutical packaging sector is expected to reach USD 101 billion.1 Recognizing product codes using machine vision enables the storage and processing of package-specific manufacturing data, as well as the electronic search and extraction of codes and dates, which is important in the development of intelligent product handling systems.\n\nCardboard is typically used in pharmaceutical product packaging. Boxes made of cardboard have curved surfaces. When accurate text recognition of text printed on surfaces is needed using 2D machine vision, the curvature is a difficulty in itself as it causes uneven illumination of the packaging surface as presented in Figure 1. Several printing methods are used to print the expiration date and manufacturing batch codes that are important for the usage and handling of pharmaceutical packages. Changes in physical conditions in package imaging as well as printing methods that generate low-contrast text and irregular letter forms make it more difficult to recognize these codes. The recognition process is especially challenging because there are many variants in the codes used on various packages using different printing methods, as well as variances in the codes’ forms, structures, regularity, and colors. In the past ten years, product code recognition techniques have advanced, and several researchers have become more interested in this area of study.\n\nThe purpose of this research is to demonstrate that, despite field difficulties, texts with imperfections printed on pharmaceutical packaging can be effectively recognized using appropriate pre-processing and deep learning. In the experimental part of the study, the recognition accuracy of the text recognition method trained on a real-life image set and the number of unrecognized characters is measured using generally comparable metrics, and the novel deep learning text recognition method is compared to the Tesseract OCR method in the recognition of dot matrix and ink-printed characters using four multi-object recognition metrics. It is important to note that information related to expiration dates and manufacturing batch codes on pharmaceutical packages is important for safety and must be accurate and reliable. The rest of the paper is organized as follows: we discusses the related works from a survey of the literature. A description of the proposed methodology is then presented. Analysis of experimental results is then presented and finally, the conclusion and future works are presented in the final section.\n\n\nRelated works\n\nThe product’s manufacturing markings are recognized by a computer and optical character recognition software from the digitized image produced by an imaging system, which is based on the amount of light energy reflected from the surfaces of the objects in the scene.2 Optical Character Recognition (OCR) methods that compared groups of pixels detected on the surface of a product to models given to the system have been increasingly overtaken by methods utilizing deep learning technology.2 OCR software can recognize characters that are regularly shaped and have good contrast against a simple background.3 However, the difficulties in recognizing packaging texts, which are described with solutions,2 reduce the usability of the OCR method in this high-accuracy task.\n\nTesseract OCR is a widely used open-source optical character recognition program. It has been in use since 1985 and has evolved significantly throughout the years. Over time, Tesseract OCR has changed. Numerous languages are supported, image binarization is included, and Tesseract 4 added an OCR engine with an LSTM-based neural network, which process an input image line by line into boxes and then feeds them to the LSTM network, which produces the recognition result.4\n\nOCR text recognition was used in the research by Gong et al. (2020) to recognize text in images of food packaging. The text that was printed on the product package had slanted characters. Some images were light-exposed, and some texts were printed with poor print quality. The Tesseract OCR method correctly recognizes 31.1% of characters.5 In the same study, the deep learning text recognition method achieves 95.4% recognition accuracy with identical data.\n\nDeep neural networks are being used more frequently for product text recognition as of 2018, which has made it possible to recognize inconsistent characters, detect and recognize the text of various sizes in images taken in real-world conditions, and even recognize texts from moving packaging.2 Before that, it appears that conventional recognition methods were more frequently employed.2\n\n\nMethods\n\nThis research focused on utilizing deep learning to recognize texts printed on the surface of differently curved packages. Dot matrix printed texts and pressed ink-marked texts are the focus of our study. As part of our research work, we have developed a novel algorithm for recognizing text from images taken from packages. The source images are taken in a controlled imaging environment and binarized with a method in Ref. 6, which, despite the variable contrast between the text and the background and the curvature of the packaging surface, is capable of accurate and robust binarization. The novel method’s text recognition ability is first tested in terms of text recognition accuracy and the number of unrecognized characters. Furthermore, using four criteria, the performance of the deep learning network constructed for text recognition is compared against Tesseract OCR. Text recognition of texts pressed without ink, stamped, or laser printed is excluded from the study. In the experimental recognition tests, we used a set of images of a Finnish health technology company taken from real medical packages, of which 16 pcs were with dot matrix printed texts, and 11 pcs with pressed ink-marked texts. The study’s text recognition was performed on the packaging production batch and expiration date markings, and titles. An example of the binarized images used for text recognition is shown in Figure 2. In the experimental tests, real pharmaceutical packages have been used to ensure that the results of our research are reliable. Since the text recognition of pharmaceutical packages is a new area of research, the number of source images was limited. However, this limitation is only temporary, and research will continue as more source images become available.\n\nDeep Learning (DL) is a subset of machine learning in which neural networks are used to learn from data and generate predictions or choices. It is described as a method of teaching a computer to learn from data and make predictions or judgments using neural networks, much like a child does. Deep Learning can be used for a variety of tasks, including image classification, speech recognition, and natural language processing.\n\nRegions with Convolutional Neural Networks (R-CNN) architecture combines rectangular region proposals with convolutional neural network features. Figure 3 represents the R-CNN architecture in character recognition. For character recognition, R-CNN first extracts several object-independent region proposals from the source image using the method of selective search (Figure 3a). Each region is sent on to the convolution neural network, for the creation of a fixed-size feature vector corresponding to them (Figure 3b). The feature vector is then used as the input to a set of linear SVMs that are trained for each class and output a classification (Figure 3c). To obtain the most accurate coordinates, the vector is also fed into a bounding box refinement layer of a network (Figure 3d).7,8\n\nThe dataset contains 27 binarized source images, 16 of which have dot matrix texts and 11 of which have pressed, and ink-marked texts. The network was trained using the stochastic gradient descent with a momentum optimization algorithm, with a learning rate of 1e-4 that is reduced by 0.02 after every eighth epoch. Other network training parameters include max epoch 400, and a mini-batch size 58. A pre-trained Alex-Net network with transfer learning is combined with a Region-CNN deep learning network to recognize the 43 different categories of objects in the images. First, images are fed into Alex Net CNN (i.e., 227×227×3). The dataset is divided into two parts: training and validation. The final layers of the pre-trained Alex Net CNN were modified to match the number of classes in the training dataset. To prevent network overfitting, data augmentation was used for training images. Modified Region-CNN was trained using the augmented training images and labels.\n\n\nResults\n\nThe experiments aimed to evaluate the recognition accuracy of the deep learning method for text recognition on real-world pharmaceutical packages. The experiments were carried out on available dot matrix, printed, and ink-marked texts. The recognition results were achieved by comparing the recognition results obtained with the test image set to the ground truth data.\n\nThe accuracy of the text recognition of the novel model was analyzed in the first test with a dataset using a generally comparable evaluate Detection Precision9 function. The function outputs the accuracy of each object class for a multi-object detector. Also, the evaluate Detection Miss rate10 function was used in the evaluation, calculating the model’s performance based on the number of undetected objects in the recognition of multiple objects. Figure 4 shows the mean Average Precision- and log Average Miss Rate-curves plotted for all object categories in the character recognition test data set.\n\nRecall and precision are cell arrays for a multi-object detector, with each cell containing data points for each object category. Log Average Miss Rate (LAMR) is also calculated for each object category separately. The mean Average Precision (mAP) metric was used to evaluate the performance of the multi-object detection model. The mAP is calculated by taking the mean of the AP values for each object category yielding a Precision value of 0.96 and a Recall value of 0.72. The detector’s performance findings demonstrate the percentages of accurate classification (precision), and all searchable objects detected (recall). The mAP rating of 0.72 indicates that the detector performed well, produced accurate findings, and found a large percentage of the objects analyzed. The Log Average Miss Rate (LAMR) function was used to analyze the performance of the multi-object recognition task. The measured LAMR value of 0.13 demonstrates that the number of undetected objects was very low, and that the detector performed well in the multi-object detection test.\n\nIn the second experiment, the same data set with images containing text was given for text recognition to both the novel deep learning model and Tesseract OCR. The numbers of all characters for each image were first counted. The number of correctly identified and incorrectly recognized characters, as well as the number of unrecognized and double-identified characters, are calculated for performance evaluation. This gave detailed information on the methods’ text recognition accuracy. The threshold value (α) of the R-CNN model in the recognition accuracy test was set to 0.99.\n\nIoU (Intersection over Union) is a common object detection evaluation metric. IoU is a measure of the intersection being the overlap between the ground truth boundary box and the algorithm-detected boundary box.11 IoU, as depicted in Equation (1), is calculated as the area of overlap between the ground truth bounding box and the predicted bounding box divided by the area of the union of these two.11\n\nPositive and negative samples are required for all supervised learning methods. Using a human face detector as an example, images with faces are positive samples, whereas images without faces are negative.12 After testing, a group of positive and negative samples can be classified into one of four states, as shown in Table 1.12\n\nPositive samples may create the following scenarios:\n\n1. TP (true positive), in which a positive sample is determined as the target by the detector, IoU ≥ α.\n\n2. FN (false negative), in which a positive sample is determined by the detector to be non-target.\n\nNegative samples can create the following scenarios:\n\n3. TN (true negative), in which a negative sample is determined by the detector to be non-target.\n\n4. The detector determines FP (false positive), or a negative sample, as the target, IoU < α.\n\nThese formulas are used to calculate precision and recall:\n\nPrecision is the proportion of all correctly retrieved samples (TP) that account for all retrieved samples (TP + FP).12 Its equation is, as given in (2):\n\nThe recall is the proportion of all correctly retrieved samples (TP) that accounts for all objects that should be retrieved (TP + FN).12 Its equation is, as given in (3):\n\nAccuracy is the proportion of all correctly retrieved samples that account for all samples.12 Its equation is, as given in (4) as follows:\n\nThe F-Measure, as depicted in Eq (5), is the weighted harmonic average of precision and recall.12\n\nUsing these four different metrics the text recognition performance of the novel deep learning and Tesseract OCR algorithms are compared in Table 2.\n\nThe comparison test results demonstrate that the developed deep learning character recognition accuracy outperforms the OCR method significantly. The deep learning model had a recognition precision of 91.1%, while the OCR method had a recognition precision of 38.3%. The novel deep learning model was significantly more precise and efficient than the OCR method in recognizing the test set’s texts. The deep learning model has a recall value of 72.7%, while the Tesseract method has a recall value of 61.3%. When compared to the Tesseract OCR approach, the novel model’s recall value in text recognition of the target test set is 11,4% higher. On the test image set, the deep learning model’s text recognition accuracy was 69.9%, whereas the Tesseract method’s text recognition accuracy was 30,8%. The F-Measure is a number that measures the object detector’s precision-to-recall ratio. Precision describes the proportion of correct recognitions made out of all retrieved recognitions, whereas recall refers to the proportion of correct recognitions accounts for all recognition’s should have been retrieved. The F-Measure combines these two results into a numerical result that describes the object detector’s overall performance. The F-Measure of the novel deep learning model is 80.9%, which is higher than the F value of the Tesseract OCR text recognition method, which is 47.1%. This shows that the deep learning model outperforms in terms of both Precision and Recall. The results demonstrate that the novel deep learning model is a more efficient text recognition method and can recognize characters in test images more consistently. The text recognition results using the novel Region-CNN method are shown in Figure 5.\n\nLeft: recognized printed ink-marked packaging text. Right: recognized dot matrix printed packaging text.\n\n\nConclusions\n\nIn this paper, deep learning-based text recognition of pharmaceutical packages has been studied, and our novel method for expiration date and batch codes text recognition was presented. The recognition results achieved by a novel method, which is based on a regional convolution neural network, appear to be excellent and significantly outperform those obtained by Tesseract OCR, allowing the recognition of texts with inconsistencies in character shapes printed on curved surfaces with proper preprocessing.\n\nVarious methods for recognizing text printed on products have been developed. Commercial OCR engines are available that can recognize high-contrast regularly shaped texts printed on flat surfaces. In the industry, large amounts of perishable food and pharmaceutical packages are produced daily, and the texts printed on their surface is read by people several times during the operation. Text is printed on product surfaces using various printing methods to achieve cost-effective production. The pharmaceutical industry employs various printing methods, such as laser printing, pressed without ink, and pressed with ink, resulting consistent character shapes. However, characters printed using dot matrix and manual stamping methods may have inconsistencies in their character shapes. In places where pharmaceutical product packaging is handled, such as pharmacies and drug storage facilities, where it is essential to recognize texts printed with various methods, a targeted method is required.\n\nWe compare the text recognition performance obtained using a Tesseract OCR and our novel targeted method with real-life pharmaceutical packaging in this study. We find that the novel deep learning method produces highly accurate recognition results that completely outperform the results obtained using a Tesseract OCR, indicating the limitations of optical text recognition for the research’s text recognition needs.\n\nAlthough text recognition for pharmaceutical packaging texts printed with different methods is an essential task, it is still in its early stages due to the limited availability of source images of actual pharmaceutical packages for research purposes.\n\nTo the best of our knowledge, this is the first study in which deep learning is used to recognize inconsistencies in texts printed using different printing methods. We are confident that with more training data, the proposed method’s performance would increase even more.\n\nIn the following study, we focus on the domain-specific contextual processing of recognized text characters. This results in a three-phase text recognition pipeline targeted at recognizing manufacturing markings on pharmaceutical packaging. With this approach, we improve the understanding of how deep learning can be applied in this field.", "appendix": "Data availability\n\nOpen Scientific Framework: The underlying data for A novel deep learning method for recognizing texts printed with multiple different printing methods, https://doi.org/10.17605/OSF.IO/MP3RB. 13\n\nThis project contains the following underlying data:\n\n• Novel text recognition methods source code for the Octave software.\n\n• RCNN and OCR recognition methods metrics spreadsheet.xlsx (An Excel spreadsheet containing the achieved recognition results).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nGlobal Packaging Market Size. 2020.Reference Source\n\nKoponen J, Haataja K, Toivanen P: Recent advancements in machine vision methods for product code recognition: A systematic review. F1000Res. 2022; 11(1099): 1099. Publisher Full Text\n\nAlthobaiti H, Lu C: A survey on Arabic optical character recognition and an isolated handwritten Arabic character recognition algorithm using encoded freeman chain code. Paper presented at the 2017 51st Annual Conference on Information Sciences and Systems, CISS 2017. 2017.\n\nTesseract User Manual\|tessdoc (tesseract-ocr.github.io).\n\nGong L, Thota M, Yu M, et al.: A novel unified deep neural networks methodology for use by date recognition in retail food package image. SIViP. 2020; 15(3): 449–457. Publisher Full Text\n\nKoponen J, Haataja K, Toivanen P: Text Recognition of Cardboard Pharmaceutical Packages by Utilizing Machine Vision. Electronic Imaging. 2021; 33: 235-1–235-7. Publisher Full Text\n\nWang Y, Liu X, Tang Z: An R-CNN based method to localize speech balloons in comics. MultiMedia Modeling: 22nd International Conference, MMM 2016, Miami, FL, USA, January 4-6, 2016, Proceedings, Part I 22. 2016; pp. 444–453.\n\nGirshick R, Donahue J, Darrell T, et al.: Rich feature hierarchies for accurate object detection and semantic segmentation. Proceedings of the 2014 Conference on Computer Vision and Pattern Recognition. 2014.\n\nMathworks Inc: Precision Detection. Accessed 30 January 2023. Reference Source\n\nMathworks Inc: MissRate Detection. Accessed 30 November 2023. Reference Source\n\nNachappa CH, Rani NS, Pati PB, et al.: Adaptive dewarping of severely warped camera-captured document images based on document map generation. Int. J. Doc. Anal. Recognit. 2023; 1–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGong S, Liu C, Ji Y, et al.: Advanced image and video processing using MATLAB. Vol. 12. . Springer; 2018; p. 581.\n\nKoponen J: Recognition results of a Novel deep learning method for recognizing texts printed with multiple different printing methods.13 March 2023. Publisher Full Text" }	[ { "id": "174859", "date": "12 Dec 2023", "name": "Asghar Ali Chandio", "expertise": [ "Reviewer Expertise I am a PhD in Computer Vision and Image Processing. My area of research is related to text detection and recognition from natural scene images", "document analysis and OCR." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 worked on the text recognition from pharmaceutical images. This is a complex and challenges problem, however, the methods used by the authors are not state-of-the-art. Moreover, the comparison is not made with the related work.\n1. The paper lacks the novelty of the work. 2. A comparison has been made between a CNN model and Tesseract OCR, however, the Tesseract OCR works for scanned text documents. The authors should compare their work with text recognition from images (synthetic or natural scenes) methods. 3. The authors may use instance or semantic segmentation, which will give better accuracy than the RCNN. 4. Why the Recall and Accuracy values are very smaller than the Precision? 5. The Alex-Net is a very old model and is not preferred nowadays. The authors may use a state-of-the-art pretrained model. 6. The number of images in the dataset are very low. For this low data, RCNN model will not give better results. 7. There are several English grammar mistakes.\n\nIs the work 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11829", "date": "25 Jun 2024", "name": "Jarmo Koponen", "role": "Author Response", "response": "Thank you for your peer review feedback on my publication. I have revised the publication considering your comments. Here are detailed responses to the points raised: 1. The paper lacks the novelty of the work. This is valuable feedback. The title of the entire publication has been reconsidered and adapted. I have not developed the Region CNN method, but I have created a new application that uses the Region CNN method. 2. A comparison has been made between a CNN model and Tesseract OCR, however, the Tesseract OCR works for scanned text documents. The authors should compare their work with text recognition from images (synthetic or natural scenes) methods. The comparison has been performed against both a method obtained from a pharmaceutical packaging publication ( Kumar, G. P., & Prasad, P. B. (2014). Machine vision based quality control: importance in pharmaceutical Industry. International Journal of Computer Applications, 975, 8887.) and an industry-validated method. Additionally, a systematic review article ( Koponen, J., Haataja, K., & Toivanen, P. (2022). Recent advancements in machine vision methods for product code recognition: A systematic review. F1000Research, 11.) did not find an existing solution but identified a research gap regarding the state-of-the-art method in the pharmaceutical packaging field. 3. The authors may use instance or semantic segmentation, which will give better accuracy than the RCNN. Since OCR is proven to be in use in the pharmaceutical industry, we have not compared two new methods, but rather compared the new application to the previously used one. However, in a future study, we can compare this application using the Region CNN model to another application based on a different deep learning model. 4. Why the Recall and Accuracy values are very smaller than the Precision? This feedback led to a thorough review and adaptation of the performance metrics. Now, the curve surpassing all threshold values enables evaluation with three relevant metrics. 5. The Alex-Net is a very old model and is not preferred nowadays. The authors may use a state-of-the-art pretrained model. This is valuable feedback. However, although AlexNet and the Region CNN model are considered older, they contain beneficial features that are not present in newer members of the CNN family (Fast, Faster), such as a fixed size of 4096 feature vector for each region proposal. 6. The number of images in the dataset are very low. For this low data, RCNN model will not give better results. This field of study, being new, is still in the development phase. The diagram of metrics added in the paper presents insights into the model's performance. 7. There are several English grammar mistakes. I apologize for the errors. I have now corrected them. Thank you again for your valuable feedback. I have taken it into account, and it has helped improve my skills." } ] }, { "id": "201970", "date": "12 Dec 2023", "name": "Mridul Ghosh", "expertise": [ "Reviewer Expertise Artificial intelligence", "Deep learning", "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\n1. According to the title of the paper the \"novel deep-learning method for recognizing texts with multiple different printing methods\" the novelty is questionable. The authors should clearly explain the novelty. They claim they designed the R-CNN framework. The name R-CNN stands for Region-based CNN but the authors presented as region with CNN. What is the difference between them should be reflected in the paper. 2. The novelty in the framework is not seen. The authors should explain that. 3. The R-CNN framework is not explained in this paper. The authors should explain in detail. 4. The dataset section is weak. The number of images is very low. How deep-learning is working with these images though authors claim that they used data augmentation. But what are the techniques and how many images in the final dataset are not described? 5. The evaluation metrics are generally described in the experimental section before the results. 6. In the training the authors stated that they used Alexnet but it is not the R-CNN diagram. What are the requirements of these two deep learning methods? 7. In the training details the train-test rations are not mentioned. 8. The comparison with the state of the art is not performed. 9. The reference section is weak.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11828", "date": "25 Jun 2024", "name": "Jarmo Koponen", "role": "Author Response", "response": "Thank you for your peer review feedback on my publication. I have revised the publication considering your comments. Here are detailed responses to the points raised: 1. According to the title of the paper the \"novel deep-learning method for recognizing texts with multiple different printing methods\" the novelty is questionable. The authors should clearly explain the novelty. They claim they designed the R-CNN framework. The name R-CNN stands for Region-based CNN but the authors presented as region with CNN. What is the difference between them should be reflected in the paper. This is valuable feedback. I have not developed the Region CNN method, but I have created a new application that uses the Region CNN method. 2. The novelty in the framework is not seen. The authors should explain that. Referring to the previous response, I would like to add that the application is in a new area that has been scarcely studied. 3. The R-CNN framework is not explained in this paper. The authors should explain in detail. The paper has been revised to enhance the explanation of the R-CNN framework. Although the original version included an explanation through both text and an illustrative figure, we have now provided a more detailed description to clarify the framework further. 4. The dataset section is weak. The number of images is very low. How deep-learning is working with these images though authors claim that they used data augmentation. But what are the techniques and how many images in the final dataset are not described? In training details include that information. In addition underlying data section provides more information on the matter 5. The evaluation metrics are generally described in the experimental section before the results. I have thoroughly revised the evaluation metrics to ensure they are relevant to the research domain. The metrics have been updated and are now specifically tailored to better align with the study’s objectives and context. 6. In the training the authors stated that they used Alexnet but it is not the R-CNN diagram. What are the requirements of these two deep learning methods? I have clarified the requirements and differences between AlexNet and the R-CNN methods in the revised manuscript, ensuring that their respective roles and implementations are clearly explained. 7. In the training details the train-test rations are not mentioned. This information is provided both in the paper and in the 'Underlying Data' section. 8. The comparison with the state of the art is not performed. The comparison has been performed against both a method obtained from a pharmaceutical packaging publication (Kumar, G. P., & Prasad, P. B. (2014). Machine vision based quality control: importance in pharmaceutical Industry. International Journal of Computer Applications, 975, 8887.)) and an industry-validated method. Additionally, a systematic review article (Koponen, J., Haataja, K., & Toivanen, P. (2022). Recent advancements in machine vision methods for product code recognition: A systematic review. F1000Research, 11.) did not find an existing solution but identified a research gap regarding the state-of-the-art method in the pharmaceutical packaging field. 9. The reference section is weak The related work section has been strengthened, particularly in relation to the context of the study." } ] } ]	1	https://f1000research.com/articles/12-427
https://f1000research.com/articles/13-824/v1	23 Jul 24	{ "type": "Case Report", "title": "Case Report: Acquired Hemophilia by the presence of anti-factor VII autoantibodies associated with an antiphospholipid syndrome: a case report and review of literature", "authors": [ "Houssem Abida", "Bilel Arfaoui", "Nour El Houda Gueddiche", "Faida Ajili", "Sameh Sayhi", "Nadia Ben Abdelhafidh", "Bilel Arfaoui", "Nour El Houda Gueddiche", "Faida Ajili", "Sameh Sayhi", "Nadia Ben Abdelhafidh" ], "abstract": "Introduction Acquired hemophilia is a rare disease characterized by the presence of neutralizing autoantibodies against hemostasis factors, most often factor VII. However, a few cases of anti-factor VII (FVII) autoantibodies have been reported in the literature. We report a case in this regard.\n\nCase report A 28-year-old woman with no family or personal history presented with a severe hemorrhagic syndrome made of very abundant metrorrhagia with multiple compressive cervical hematomas, associated with pulmonary embolism and thrombosis. of the right external iliac vein. On the biological assessment, the patient presented with an undetectable PT with a factor VII titer less than 6%. The hemorrhagic syndrome was aggravated by infusions of activated factor VII (FVIIa) with the appearance of hemoptysis and hematomas in all four limbs. Etiological assessment concluded the presence of anti-factor VII autoantibodies as well as circulating lupus anticoagulant. Other antiphospholipid syndrome (APS) antibodies as well as antibodies against other hemostasis factors were absent. A treatment combining corticosteroid therapy (1mg per kg per day of prednisone) and Mycophenolate mofetil (MMF) (3g) was initiated. Faced with the persistence of the hemorrhagic syndrome, rituximab was administered according to the rheumatological protocol (1g on D1 and 1g on D15). Metrorrhagia improved initially but recurred with the resumption of menstruation with a drop in hemoglobin from 9 to 7 g/dl. Plasmapheresis sessions were attempted but thromboses on the catheters occurred each time. Immunoglobulin (IVIg) courses were administered. No anticoagulant was administered. TP increased to 23%. Levels of neutralizing antibodies fell from 512 to 2 IBU at the last follow-up at five months from the bleeding event. The circulating lupus anticoagulant was present on the follow-up test after 12 weeks, thus confirming the diagnosis of APS.\n\nConclusion Our patient presents an extremely rare case of acquired haemophilia. The combination of corticosteroid therapy, conventional and biological immunosupressives, IVIG and plasmapheresis saved the patient. In the absence of consensus on the treatment, it remains adapted according to the severity of the haemorrhagic syndrome and the associated comorbidities.", "keywords": [ "Antiphospholipid syndrome", "Acquired hemophilia", "immunosupressive therapy" ], "content": "Introduction\n\nAcquired hemophilia, a rare condition, is characterized by the presence of neutralizing autoantibodies against most often Factor VIII (FVIII) and in rare cases, Factor VII (FVII). These antibodies effectively inhibit the activated form of FVII by binding to its light chain. This binding disrupts the interaction between FVIIa and tissue factor (TF). The exact mechanism responsible for the development of this condition is poorly understood. Some researchers propose a paraneoplastic context, while others suggest a link to infections and autoimmune diseases, particularly Antiphospholipid syndrome (APS).\n\nIn this case, we present a new instance of an inhibitor to FVII in a young woman experiencing a severe hemorrhagic syndrome. Notably, the symptoms were exacerbated by the administration of recombinant factor VII, and the patient had a history of APS.\n\n\nCase report\n\nIn October 2022, a previously healthy 28-year-old woman presented with sudden left elbow hemarthrosis and spontaneous gingival bleeding. She had no prior personal or family history of abnormal bleeding. Upon admission, her hemoglobin concentration and platelet count were normal, but her prothrombin titer (PT) was low (11%, normal range: 70-100%), and her INR was high (7.1, normal range: 0.8-1.2). The baseline factor VII level was 6%, while the levels of other factors were normal. The patient received 5 mg of recombinant factor VII (rFVII) and was discharged the next day.\n\nThree weeks later, the patient returned to the emergency department experiencing dyspnea and dysphagia for two days. Physical examination revealed a right cervical hematoma. A cervical computerized tomography (CT) scan without contrast showed bleeding in the retropharyngeal space, causing compression of the airways and digestive tract. Her hemoglobin concentration was 10 g/dL, and PT was still at 11%. She was admitted to the hospital and received 5 mg of rFVII. The following day, the size of the cervical hematoma increased and became bilateral. The patient developed hemoptoic sputum and ecchymosis on the right arm. PT decreased to 6%. The treatment with rFVII was resumed at a higher dose (15 mg per kg every 6 hours), and two units of red blood cells were transfused. However, the hemorrhagic syndrome worsened the next day, with heavy metrorrhagia and multiple ecchymosis on both upper and lower limbs.\n\nA second CT scan revealed an extension of the retropharyngeal hematoma into the posterior mediastinum and retroperitoneal area, along with pericardial and pleural effusion. Treatment with tranexamic acid (1 g every 6 hours) and blood transfusion were initiated. The PT was undetectable this time, indicating the presence of an inhibitor to factor VII. The antibody titer was quantified as 428 Bethesda units (BU). Lupus anticoagulant was also detected, but other Antiphospholipid syndrome (APS) antibodies and antinuclear antibodies (ANA) were negative. The patient started treatment with methylprednisolone (1 g per day) and mycophenolate mofetil (3 g per day). Bleeding stopped within 24 hours, but the PT remained undetectable.\n\nA week later, the patient developed deep vein thrombosis (DVT) in the left lower limb, and a bilateral segmental pulmonary embolism was incidentally discovered during a follow-up CT scan. Enoxaparin (2000 IU every 12 hours) was initiated for anticoagulation. One month after admission, the patient experienced heavy metrorrhagia again, and her hemoglobin concentration dropped to 8 g/dL. The FVII antibody titer increased to 542 IUB. Anticoagulation was discontinued, and the patient continued taking prednisone (1 mg per kg per day) and mycophenolate mofetil (3 g per day). Rituximab infusions were administered (1 g on day 1 and 1 g on day 14), which stabilized the genital bleeding. The PT increased to 8%, and the FVII antibody titer decreased to 415 BU (Figure 1).\n\nTwo weeks later, the patient experienced heavy menstruation with a decreased hemoglobin concentration of 7 g/dL. Plasmapheresis was attempted, but only one session was successful, as the patient developed local catheter thrombosis during subsequent sessions. Intravenous Immunoglobulin (IVIg) therapy (0.4 g per kg per day) was administered for four days, leading to the cessation of bleeding by the third day. PT increased to 9%, and FVII antibody titer decreased to 62 BU. The patient’s prednisone dose was gradually reduced by 5 mg per week. Antibody titers continued to decrease to 23.5 and then 18.2 BU. A control test after two and a half months showed the presence of lupus anticoagulant.\n\nAfter a 90-day hospitalization, the patient was discharged in April 2023. She was prescribed a daily dose of 15 mg of prednisone and 2 g per day of mycophenolate mofetil. No anticoagulation therapy was recommended. Since her discharge, she has not experienced any bleeding. Her menstruations have resumed with a normal flow and duration. Her hemoglobin concentration was 10 g/dL, PT was at 23%, and the FVII antibody titer was 2 BU.\n\n\nDiscussion\n\nAcquired inhibitors of coagulation are rare but severe conditions. Among them, inhibitors to factor VIII (acquired hemophilia A) are the most common, and management recommendations have been established. On the other hand, factor VII inhibitors are rarer and less well-known. These autoantibodies can be isolated or associated with various diseases. The first case report of factor VII deficiency due to the presence of its inhibitor dates back to 1980, as a paraneoplastic syndrome associated with bronchogenic carcinoma.1 Few other similar cases associated with malignancies have been reported afterward. This condition has also been found in the context of infections, mostly HIV-related.2 It has also been described in association with autoimmune diseases, mainly APS.3 Additionally, it has been reported as secondary to drug administration, particularly penicillins.4 Therefore, when encountering acquired hemophilia, thorough screening for these associated diseases is necessary before concluding its idiopathic or isolated nature.\n\nClinical presentations of factor VII inhibitor cases seem to be correlated with antibody titers. Severe hemorrhagic syndromes and mortality have been observed with titers above 5 Bethesda units (BU).5 To our knowledge, the titer in our report is the highest reported so far. However, the presence of neutralizing antibodies has been noted in some populations, such as patients with APS, without any clinical manifestation, making it uncertain to establish a clinico-biological correlation.6\n\nUnfortunately, treatment strategies and objectives have not been clearly defined yet. For the symptomatic management of acute bleeding, procoagulant agents like tranexamic acid have been used in some cases.7 In our case, the use of tranexamic acid was not possible due to the patient’s thrombotic complications related to APS. Another particularity of our case is the possible role of recombinant factor VIIa (rFVIIa) in maintaining and stimulating the autoimmune process leading to the synthesis of these antibodies. Although not used in previous cases, rFVIIa has been suggested as a possible option for severe cases.8\n\nFor therapeutic options, the most common first-line choice has been a combination of glucocorticoids (GC) and immunosuppressive agents, with successful results in some cases.9 The choice of the immunosuppressive agent should take into consideration associated comorbidities to target both if possible. Cyclophosphamide and rituximab (RTX) have been commonly used. RTX has been employed for both remission induction and maintenance.10 To the best of our knowledge, this is the first reported case of a factor VII inhibitor treated with mycophenolate mofetil (MMF).11 For severe acute hemorrhages and refractory forms, intravenous immunoglobulin (IVIg) has been used with good outcomes.12 Lastly, plasmapheresis can be a potential alternative for life-threatening presentations. However, it is preferably performed before the administration of immunosuppressive drugs or IVIg to avoid increasing their clearance and thereby diminishing their pharmacological action.13\n\n\nConclusion\n\nIn this case report, we describe a successful treatment regimen for a severe and biologically challenging presentation of acquired factor VII inhibitor. Our approach relied on a combination of conventional immunosuppression, immunotherapy, intravenous immunoglobulin (IVIg), and plasmapheresis (PE). By employing this comprehensive treatment strategy, we effectively managed the patient’s condition and achieved positive outcomes. This highlights the importance of a multidimensional approach in the management of severe acquired factor VII inhibitors, considering both the clinical and biological aspects of the condition.\n\n\nEthics and consent\n\nSigned informed consent was obtained from the patient regarding the use of patient information for the purposes of writing a case report publication.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nDeschiens MA, et al.: Autoantibodies to factor VII in a case of acquired haemophilia. Br. J. Haematol. 1980 Feb; 44(2): 319–327. Publisher Full Text\n\nCollins PW, et al.: Acquired haemophilia in association with HIV infection: a report of two cases. Br. J. Haematol. 1992 Nov; 80(3): 126–127. Publisher Full Text\n\nKnoebl P: Acquired inhibitors of coagulation. Semin. Thromb. Hemost. 2012 Feb; 38(1): 01–04. Publisher Full Text\n\nSuárez-Cortina L, et al.: Acquired hemophilia A associated with penicillin allergy: a case report. Ann. Allergy Asthma Immunol. 2016 Oct; 117(4): 264–272.e4. Publisher Full Text\n\nLacroix-Desmazes S, et al.: Acquired factor VII deficiency associated with autoimmune disorders: two case reports and literature review. Haemophilia. 2015 Nov; 22(6): 268–275. Publisher Full Text\n\nJohnson EA, et al.: Factor VII inhibitor presenting as a life-threatening hemorrhage during pregnancy: a case report. Obstet. Med. 2019 Sep; 12(3): 157–160. Publisher Full Text\n\nSmith JM, et al.: Acquired factor VII deficiency: a case report and review of the literature. Blood Coagul. Fibrinolysis. 2020 Jul; 31(5): 324–329. Publisher Full Text\n\nLee G, et al.: A rare case of acquired factor VII inhibitor presenting with severe bleeding: a case report. BMC Hematol. 2022 Jan 21; 22(1): 1. Publisher Full Text\n\nFranchini M, et al.: Successful treatment of acquired factor VII inhibitor with cyclophosphamide and corticosteroids. Ann. Hematol. 2004 Nov; 83(11): 720–722. Publisher Full Text\n\nBoggio LN, et al.: Successful treatment of acquired factor VII deficiency with glucocorticoids and rituximab: a case report and review of the literature. J. Pediatr. Hematol. Oncol. 2013 Jul; 35(5): e203–e205. Publisher Full Text\n\nBachoud-Levi AC, et al.: Mycophenolate mofetil for acquired factor VII inhibitor. Ann. Hematol. 2002 May; 81(5): 288–289. Publisher Full Text\n\nPiovella F, et al.: Intravenous immunoglobulin as first-line therapy in acquired factor VII deficiency. Blood Coagul. Fibrinolysis. 2018 Oct; 30(7): 246–248. Publisher Full Text\n\nGiangrande PL, et al.: Plasmapheresis in acquired factor VII deficiency associated with lupus anticoagulant. Br. J. Haematol. 1994 Jan; 87(1): 148–152. Publisher Full Text" }	[ { "id": "332507", "date": "13 Nov 2024", "name": "Mark Schreuder", "expertise": [ "Reviewer Expertise My area of research is in coagulation factors and bleeding diseases", "including hemophilia." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, Dr. Houssem and co-authors present a rare instance of acquired hemophilia resulting from factor VII autoantibodies. The patient’s biological assessment also showed persistent lupus anticoagulant, indicating an underlying antiphospholipid syndrome. The authors detail the successful treatment of the patient with a regimen that included corticosteroids, immunosuppressants, immunoglobulin therapy, and plasmapheresis. This case is both intriguing and thoroughly described, providing valuable insights into the management of such complex conditions.\nI only have a few minor comments:\nIn the abstract, the authors wrote that “Acquired hemophilia is a rare disease characterized by the presence of neutralizing autoantibodies against hemostasis factors, most often factor VII”. I assume the authors mean factor VIII. It would be helpful to add y-axis descriptions and units in figure 1 to better visualize which axis belongs to which curve and to understand what the curve represents. In addition to the previous comment, it would be helpful to mention in the text or in the figure caption what point in time represents the start of the figure 1 curves. As I understand, the PT was first measured in October 2022 and was subsequently tested in further episodes. The BU was measured first about three weeks after the patient was presented to the hospital, which would be October/November 2022. Yet the figure 1 curves start in December 2022.\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/13-824
https://f1000research.com/articles/12-1214/v2	29 Sep 23	{ "type": "Research Article", "title": "In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease", "authors": [ "Lazuardi Mochamad", "Selvaraja Malarvili", "Khairat Jasmine", "Vuanghao Lim", "Selvaraja Malarvili", "Khairat Jasmine", "Vuanghao Lim" ], "abstract": "Background: Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods: This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulency titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) and in chicken kidney cell culture. Results: Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions: QLCs from flavonoids from the leaves of BD have antiviral bioactivity against NDVs and may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.", "keywords": [ "Antiviral agents", "Benalu Duku", "Health life", "Herbal medicine", "Inactivant" ], "content": "Introduction\n\nMistletoe plants contain flavonoids; these compounds are often found in the leaves. The mistletoe Dendrophthoe pentandra (L.) Miq., known in Indonesia as Benalu Duku (BD), has multiple benefits, especially for medicines intended to treat animal and human diseases.1,2 Interestingly, reports stated that the leaf part of BD comprises mainly flavonoid elements with a polyphenol structure or analogous to quercetin, and are therefore expected to have antimicrobial potential.3 Consequently, they may be antibacterial or antiviral compounds; thus, they show great promise for further development into candidates for new plant-based, or specifically mistletoe-based, antimicrobial compounds.4 In terms of its antiviral properties, there are generally three main places where functional groups may cause these effects, namely complex aromatic structures, electron attractor groups, and quercetin analogs.5,6 That concept can be explained using theory based on quantification structure-activity relationship versus virus agents. As an anti-virus, it is suitable for killing viruses that live and develop in host eukaryote cells that have the property of being able to regrow themselves against eukaryote cells that have been damaged such as intestinal cells, skin epithelial cells, epithelial cells of the reproductive tract.7 This phenomenon can be more easily determined after initial in vitro screening in a group of viruses under aerobic conditions or living in hydrophilic cells, such as a laryngotracheal cell.8 These compounds are said to be quercetin analogs because the bioactive separation of quercetin in plants is difficult and frequently contaminated with other compounds.9 Thus, the main chemical and physical characteristics of the quercetin structure are retained in quercetin-like compounds (QLCs).\n\nSome viruses can survive in laryngotracheal cells; one such virus is the Newcastle disease virus (NDV) that often affects poultry, especially chicken and birds. Infected cases primarily present as acute respiratory symptoms, then depression, and finally as nervous manifestations in late infection.10 Diarrhea may be the predominant clinical symptom.11 The severity of the infection is determined by the virulence of the infecting virus and the host susceptibility.11,12 The occurrence of cases is reportable; they may result in trade restrictions. NDV is also known as avian paramyxovirus serotype 1 (PMV-1). NDV is an RNA virus with at least 11 pathogenic PMV serotypes in poultry.13 The classification of NDV isolates is divided into three groups according to virulence: the malignant group (velogenic), the moderately malignant group (mesogenic), and the group with low malignancy (lentogenic).14\n\nThe exploration of QLCs as an antimicrobial agent, especially an antiviral, is critical given the demands of the World Health Organization (WHO) and Food Agriculture Organization (FAO) regarding the third sustainable development goal (to ensure healthy lives and promote well-being for all at all ages) will be related to veterinary drug residues and antimicrobial resistance (AMR).14,15 The selection of natural compounds from plants, such as QLCs, will minimize the risk of the adverse impacts of AMR and animal drug residues in livestock that will be consumed by both adults and children.16,17 It is known that efforts to replace bioactive antivirals with synthetic compounds will impact the problem of veterinary drug residues; thus, efforts to use QLCs as a Remedies Cardinal formula for an antiviral drug are very useful.18,19 The purpose of the study was to prove the ability of QLCs extracted from BD leaves to eliminate the NDVs that cause outbreaks in poultry. In this study, in vitro investigations were performed to determine the antiviral potential of bioactive compounds in BD leaves. The preliminary data produced will be continued with in vivo studies or field tests using bioactive formulations containing QLCs in the future.\n\n\nMethods\n\nBD plants were obtained from the south of Sumatera, in the district of Muara Enim, Indonesia, at the geographical coordinates of 3° 39′ 0″ south, 103° 48′ 0″ east. The plant, as mistletoe, is Dendrophthoe pentandra (L.) Miq., was identified by the Directorate Control of the Natural Science Collection, Indonesian Consortium Research and Innovation National, official letter number B-1679/11.6.2/DI.05.07/6/2022, at the address of Raya Jakarta – Bogor Km 46 on June 7th, 2022, with the Indonesian name BD. The collection of the samples started in March 2022 and continued until August 2022.\n\nThe virus was identified as a NDV obtained from an infected chicken at a chicken breeding farm in Gresik District (East Java), Indonesia, in October 2022 (at the geographical coordinates of 7° 9′ 14″ south and 112° 39′ 22″ east). The seed virus was characterized as velogenic by referring to the veterinarian officer (VO) of that breeder and agreed upon by the VO and the head of the research team. The VO performed identification using a modified field test model based on live virus vaccination. A total of 30 male chickens aged 6 months were divided into six groups, and each group consisted of five chickens. Group 1 was given the name without vaccination (NV), group 2 was given the name vaccination Avian Encephalitis (AE), group 3 was named the vaccination infectious avian influenza (AI) vaccine, group 4 was given the name vaccination Infectious Laryngotracheitis (ILT), group 5 was given the name vaccination infectious bronchitis (IB), and group 6 was given the name vaccination Newcastle disease virus (NDV). Of the six groups, it turned out that the NV, AE, AI, ILT, and IB groups were infected with the NDV virus, with symptoms such as the head being often positioned downward towards the feet, drooling frequently, no appetite, pale eye mucosa, and body temperatures reaching 41oC. During the auscultation, there was noise and the sound of secretions. The group that had received the NDV vaccine was not infected with the virus. From the five NV groups, saliva was collected and made free from bacterial fungi and parasites after being added to antibiotics (Penicillin G w/v Oxoid Pharma 1.500.000 UI catalog number: CT0043B; Kanamycin Injection Meiji Pharma v/v 50 mg/mL), anti-fungi (Nystatin Taisho Pharma w/v 100.000 IU), and anti-parasites (Metronidazole Medicina Interna Pharma w/v 50 mg/mL). Substances of NDV were performed as velogenic substances and characterized as follows; (a) pyrogen-free, (b) iso-tonic, (c) iso-ionic, and (d) iso-hydric.20 The NDV substances were propagated using chicken embryos in eggs (aged 4 to 5 days) by injecting 0.25 mL of the NDV substance onto the eggshell until the needle was in the air cell part of the egg. The part of the injection site was covered with paraffin solidum pharmaceutics grade (PT Nusa Kimia-Indonesia catalog number: 01-parafin padat) and incubated on a rotated interval movement model for 2 days (37°C) using eggs incubators (Mesin Penetas Surya Rejeki-Indonesia). After incubation, chicken embryos were taken, crushed, and washed using Ringer Lactate Solution (RLS Meiji Pharma). The washing was repeated up to three times, and centrifugation (3000 rpm, 30 min) was performed to obtain the supernatant. The supernatant was used for testing as a field virus, NDV, to be developed and propagated using Vero Cell culture in Dulbecco’s modified eagle’s medium (DMEM, Merck catalog number: D5030) added to 10% of Fetal Bovine Serum (FBS, Merck catalog number: 12003C). The substance of NDV was propagated into the Roux’s plastic bottle (250 mL contains 14×105 Vero cells infected/mL), then harvested to obtain the virulence level of 10−5 Tissue Culture Infectious Dose 50% (TCID50).\n\nApproximately 5 kg of BD as samples were washed using aqua demineralization (PT Brataco Surabaya-Indonesia catalog number: 01-Aquadem) and separated from other leaves to be pulverized using a pulverized apparatus (HBS Blender catalog number: MTA-94200747) so that the powder obtained weighed approximately 284 g. Pulverized BD was extracted by methanol pro analysis (p.a.) grade (Merck catalog number: 111457-85-3) using a movement maceration method for 48 h. The obtained raffinates were further concentrated using a rotary evaporation device (Buchi Rotavapor R-200 Rotary Evaporator) connected to a water bath apparatus adjusted at 40oC. Crude extract from Raffinate from methanol p.a. was added to sterile aqua demineralization (100 mL) and put in a partition separator flask (Iwaki Separator Flask 250 mL catalog number: 6401FS100). Further, ethyl acetate p.a. grade (Merck catalog number: 141-78-6) was added up to the top volume level. The results of tannin-free raffinate were further dried using nitrogen vapor in a water bath at 40oC (Funke Gerber Germany catalog number: WB 436). Thus, tannin-free raffinates were obtained. Tannin-free raffinates were further partitioned by workflow, added to the methanol p.a. grade, and repartitioned in a partition flask with n-hexane p.a. grade as a menstruum solution. The partitioned raffinate was dried using nitrogen vapor at pharmaceutical grade from PT Matesu Gotty-Surabaya (Indonesia), immersed in a water bath, and set at a temperature of 40oC.21,20\n\nThe raffinates were analyzed for flavonoids using two qualitative flavonoid test methods. In the first method, 1 mg of the obtained raffinates was randomly taken and 10 mL of methanol p.a. grade (Merck catalog number: 111457-85-3) was added until dissolved. Especially for raffinate solution, 0.6 mL of iron chloride (III) pa solution, 1% (w/v) (Merck catalog number: 7705-08-0), was added, causing a bluish color change as well as an indicator of flavonoid content in the extract. In the second method, 1 mg of dried raffinates was randomly taken and dissolved in 10 mL (w/v) methanol p.a.; then, 3 mL of the mixture was taken and placed in three 10 mL test tubes (control tube A, tube B color indicator, tube C test sample). Then, concentrated hydrochloride acid (HCl) p.a. (Merck catalog number: 109057) was prepared and 10 drops were placed in tube B and tube C. Tube B was heated by a burner for 5 min and became more concentrated. To tube C (test tube), zinc p.a. (Merck catalog number: 7440-66-6) 0.1 mg was added. The tube was left to stand for 5 min and the color change was observed in tube C compared with tube B and tube A; tube C appeared green, tube A was brown, and tube B was dark. The green discoloration of tube C indicated that flavonoids were present.\n\nIn the second method, 1 mg of the viscous extract was taken randomly and dissolved in methanol p.a., 10 mL (w/v). Quercetin dihydrate p.a. (Merck catalog number: 551600) was also prepared as a standard 1 mg dissolved in methanol p.a., 10 mL (w/v). The thin-layer chromatography (TLC) containing activated magnesium silicate was produced by Supelco Corp. (Catalog number: 1343-88-0). The lower limit was measured at 2 cm and marked as a line. Additionally, prepared in the chamber of the mobile phase solution was 1-butanol p.a. (Merck catalog number: 101990), acetic acid p.a. (Merck catalog number: 64-9-7) and sterile water (Perusahaan Terbatas Kimia Farma Indonesia catalog number: 01-aquadest) in a 1:1:1 ratio. Dropwise, samples of raffinates were added to methanol p.a. and quercetin standard on the TLC paper, and elution was performed using the prepared mobile phase. The paper was taken and observed under UV light at a wavelength of 366 nm (yellow light). The calculation of the retention factor (Rf) between the two compounds was performed to aim to produce the same Rf value. The presence of flavonoids will be more noticeable when spraying ammonium solution p.a. (Merck catalog number 119812) on post-elution TLC, both sample drip and standard, with red color appearance indicators. The red color indicates the presence of flavonoids in raffinate.20\n\nIn this stage of the work, the mobile phase was optimized by searching for the mixed fraction of the composition of the polar solution so that it could separate the analytes using the stationary phase for TLC. The mobile phase fraction sought was a mixture of water and methanol, water pro chromatograph (p.c.)–methanol p.c. so that it could later be applied to semi-preparative HPLC devices. In the search, the ratios of 4:6, 3:7, 2:8, and 1:9 were tested. The selected fraction of the mobile phase was used to separate the compounds of BD extract from impure compounds to obtain the QLCs. The implementation of the research began with the preparation of TLC and the preparation of the mobile phase solution. A mobile phase mixture of water p.c.-methanol p.c. was prepared in a chamber. TLC was outlined according to the area to be dripped, which was 2 cm from the bottom of the chamber. Furthermore, the chamber was added to the mobile phase solution with a note that the surface of the mobile phase solution does not exceed the line limit of 2 cm. Furthermore, chamber saturation was carried out by inserting filter paper and the chamber was closed followed by the dripping of raffinate (QLCs) and quercetin standards on the marked TLC. The next step was to eluate the TLC that has been dripped into the two substances for 30 minutes. The end of the elution was carried out by drying of TLC and spraying of ammonium solution. The results of the TLC were carried out Rf examination using UV TLC viewer equipment from Vilber (Spain), Lourmat TLC Viewing Chamber CN-15.LC 4121 4155 1 (at wavelengths 366 nm).\n\nDried samples, as a crude extract compound at 39 g, were added to the methanol pro chromatograph, ready for purification of the obtained flavonoid compounds or polyphenol substances (carbon-3 and carbon-6). Chemical compounds for the separation of flavonoid compounds with semi-preparative HPLC are used with high purity or pro-chromatography at a purity of 99.99% of the active substance as methyl alcohol (CH3OH) or 99.9% of the active substance, namely pro-chromatographic water (H2O). The standard for identification of the polyphenol substances was quercetin p.a. grade at 99.97% purity of active substances as 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one (C15H10O7).\n\nThe HPLC equipment used was a Shimadzu CBM-20A Communication Bus Module with a photodiode array detector in an ultraviolet–visible (UV–visible) M20A spectrometer, in which a preparative column (Xtendedlife diameter, 30 mm; length, 100 mm; particle size, 7 μm; catalog number 35007-109370XL) was fitted. The HPLC system was set to isocratic elution parameters: detection, 230–700 nm; flow rate, 0.5 mL; stop time, 20 min; mobile phase solution, 70% methanol pro chromatograph (p.c.) (Merck catalog number: 67-56-1): 30% water p.c. (Merck catalog number: 7732-18-5); loop injection capacity, 250 μL. In the second part of the experiment, the standard quercetin p.a. was dissolved (w/v) in the mobile phase solution at serial concentrations of 0.5, 0.75, and 1 μg/mL, then injected into the HPLC system in 0.5 μL aliquots. The crude raffinates from the workflow of the chromatography column were dissolved in the mobile phase solution, filtered using a 0.20 μm filter (Merck catalog number: CLS431212), and injected into the HPLC system. The sample chromatogram was compared with the standard chromatogram, quercetin p.a., based on retention time (RT), and the concentration of flavonoids in the sample was calculated based on the area of the standard chromatogram compared to the area of the sample chromatogram. In the third part, flavonoid compounds in matrix samples were isolated using analyte columns based on standard RT, and analytes from waste products were collected from HPLC equipment.21,22\n\nThe physicochemical properties of the two compounds were used to determine the analyte’s molecular structure similarity test (QLCS) in comparison to the reference molecular structure (quercetin), which was carried out using a physical-chemical-based test apparatus. The analysis of the phenomenon of adsorption–partition against the stationary phase octadecyl xylene (reverse-phase) was determined using HPLC. The HPLC specification used a similar workflow for the separation of QLCs from BD flavonoids. The column installed was an RP C18 15-cm stainless steel with a diameter of 0.20 μm (Agilent product catalog number 5982-1111). As for the mobile phase, water p.c.–methanol p.c. was used with a composition of 30:70. The flow rate was set to 0.5 mL/min and the column temperature was set to 20°C. The detector was a photodiode array with settings of 190–900 nm. Observations were made by comparing the similarity of RT between QLCs and quercetin standards.21,23,24\n\nThe analysis of QLCs compared with standards was by Fourier transform infrared (FT-IR) spectrophotometer transmission method by potassium bromide p.a. (Merck catalog number: 7758-02-3) observed using peak similarity ranging from 4000 cm−1 to 400 cm−1, percentage transmission (%T), 20 scans, at a resolution of 4 cm−1. The instrument FT-IR Spectrum One Perkin Elmer (PN: 09934358) produced by LabMakelaar Benelux B.V. Knibbelweg 18C, NL-2761, JE Zevenhuizen (ZH) (The Netherlands) was used. The research workflow for FT-IR observations was prepared in the Faculty of Pharmacy Universitas Airlangga. The implementation of FT-IR begins with the manufacture of tablets by mixing analyte QLCs with Kalium bromide (KBr) powder and mashing them in a porcelain mortar. Furthermore, it was printed into a tablet using a tablet printer device, and the formed tablet was placed on the basket of the FT-IR device and immediately checked by first setting the wavenumber according to the specifications of the FT-IR device. The technique was also carried out on a standard compound (quercetin).6,25,26\n\nProton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) were performed using a JEOL NMR (JNM ECS-400, 400 MHz). Analysis of NMR protons and NMR carbons begins by dissolving analytes (QLCs) using the solvent-deuterated methanol (CD3OD), as well as the comparison compound (quercetin). The solution made was added to the raw compound tetramethyl silane, (Si (CH 3) 4), and readings were carried out according to the specifications of proton NMR and carbon NMR devices. The spectra plotted by the JEOL show chemical shifts (δ) in parts per million (ppm) relative to the specified deuterated solvent. Spectra were calibrated using the residual solvent peaks for CD3OD (δH: 3.3 ppm; δC: 47 ppm) as appropriate. Coupling constants (J) were used quoted in Hz and were rounded to the nearest 0.1 Hz. Splitting patterns were observed abbreviated to singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m).21,27–29\n\nThe molecular mass of QLCs was determined using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) connected to UNIFI software as a process control (Waters Corporation, 34 Maple Street, Milford, MA 01757, USA). The data were kept in the system computer and library of the instrument for matching the analytes. The column installed was octadecyl xylene (ODS) C18 stainless steel (Agilent product catalog number 5982-1111). The column oven and autosampler injectors were set at 40°C and 15°C and the volume capacity injector was set at 10 μL. The running parameters were adjusted along a gradient system using the mobile phase of A (acetonitrile pro chromatograph containing 0.1% formic acid p.a.) and B (water pro chromatograph containing 0.1% formic acid p.a). The flow rate was adjusted to 0.6 mL/min. The MS parameters were adjusted to the Tof MSE mode using electrospray ionization (ESI) at +/− and an acquisition range of 50–1200 Da. The following analysis criteria were adopted: mass error reading analyte ≤ 5 ppm; isotope match m/z root-mean-square (RMS) ≤ 6 ppm; and isotope match m/z RMS % ≤ 10 %, analyte intensity ≥ 300. For one fraction, the brake was < 4 in the fragment elucidation system match.30,31\n\nChicken kidney (CK) cells were cultured from healthy leghorn chickens (age, 3 weeks; approximately body weight, 0.8–1.0 kg). The chicken was free from specific chicken diseases caused by viruses, bacterial, and fungi as observed by a veterinarian in Surabaya-Indonesia (geographical coordinates 7° 20′ 09.7″ S, 112° 46′ 15.7″E, Surabaya-Indonesia). Animal Ethical Clearance was carried out through testing the animal ethics trial team from the Faculty of Veterinary Medicine, Universitas Airlangga, which had been proposed since the research began. The exam session consists of seven examiners, all of whom are veterinarians, and were specially appointed by the Faculty of Veterinary Medicine, Universitas Airlangga, to test the research proposal. The animal ethics clearance certificate was issued with No: 1.KEH.060.04.2023 on May 12th, 2023.\n\nThe cells were prepared, maintained, and propagated at the Research Center, Apply, Development of Veterinary Pharmacy Science, Faculty of Veterinary Medicine Universitas Airlangga.32 The cells were developed and stimulated to grow in a 500 mL Roux plastic bottle in three types of medium: Eagle medium (EM), serum-free medium (SFM), and standard suspension medium (SSM). All cell culture media were sterile, free from pyrogenic substances, iso-tonic, iso-ionic, and iso-hydric.\n\nOther environmental substances used in the in vitro test and cell culture were nutrient broth p.a, (Merck catalog number: MFCD01867738), N-acetyl ethyleneimine (AEI) 0.05% (Chemsky-Shanghai International Co., Ltd., catalog number: 32344), and sterilized phosphate-buffered saline (PBS) (Merck catalog number: P4474), Tween-40 (Merck catalog number: 9005-66-7), and polyethylene glycol (PEG) (Merck catalog number: 25322-68-3). Additional chemicals needed were 0.25% Trypsin Versen solution (Merck catalog number: 9002-07-7), sodium thiosulfate solution (Merck catalog number: 7772-98-7), citric acid (Merck catalog number: 77-92-9), trypan blue (Merck catalog number: 72-57-1), FBS (Merck catalog number: 12106C), and 10% Hibitane solution (Merck catalog number: 56-95-1). Antibiotics and fungicide were also used: Penicillin G w/v Oxoid Pharma 1.500.000 UI catalog number: CT0043B; Kanamycin Injection Meiji Pharma v/v 50 mg/mL), anti-fungi (Nystatin Taisho Pharma w/v 100.000 IU). We did not use commercial EM, SFM, and SSM media as they were not able to support the growth of CK cells in our labs. Thus, the formula of the CK culture cell medium was modified and optimized.\n\nOne liter of EM medium was supplemented with sodium chloride 6.4 g (Merck catalog number: 7647-14-5), potassium chloride 400 mg (Merck catalog number 7447-40-7), Mg2SO4.7H2O 200 mg (Merck catalog number 100034-99-8), glucose 4.5 g (Merck catalog number: 492-62-6), Fe (NO3)3.9H2O 0.1% 1 mL (Merck catalog number: 7782-61-8), aquabidest 750 mL (PT Kimia Farma Surabaya catalog number: 01 Aquadest), NaPO2H2.2H2O 140 mg (Anish Chemical-India catalog number: 28351090), and phenol red 0.1% 8 mL (Merck catalog number: 143-74-8). Other substances were antibiotics (Penicillin G w/v Oxoid Pharma 1.500.000 UI catalog number: CT0043B; Kanamycin Injection Meiji Pharma v/v 50 mg/mL), anti-fungi (Nystatin Taisho Pharma w/v 100.000 IU). The final ingredients of the formal SFM were sodium chloride solution 0.424 g (Merck catalog number: 7647-14-5) and sodium hydroxycarbonate 0.33 g (Merck catalog number: 15630-89-4). The composition of SSM was the same as SFM, with the addition of 10% of FBS.\n\nCell culture was performed in a sterile biosafety cabinet (Laminar Air Flow Work Station, Sara Mechatronix) between 20°C and 23°C, as follows: one living organ was obtained and the surface membrane was cleaned and immediately inserted in PBS 25% (5–10 min) with gentle shaking to eliminate impurities.32,33 The organ was directly inserted in the barrel of the 50-mL disposable syringe by first opening the plunger. Next, the plunger was pressed to pinch the organ and it then disintegrated into fine cells out of the syringe adapter part (without the presence of a needle) and was transferred into a centrifuge tube. The cells were gently washed with PBS and the tube was centrifuged at 3000×g for 10 min (Techne Genofuge 16M Lab Benchtop Centrifuge catalog number: 75004231). The cell pellet was collected, and the supernatant was discharged. An equal amount of EM was added and whisked to the cell pellet for 10 min with a rubber head glass dropper pipette to achieve a homogenous solution. This was followed by the addition of 5 mL of Trypsin Versen, and the mixture was immediately transferred into a Roux culture bottle and incubated for 35 min (37°C). Furthermore, the addition of 10% FBS while gently shaking was to inhibit the activity of Trypsin Versen, because the binding of serum with Trypsin Versen will inhibit the work of Trypsin. Finally, 250 mL of SSM was added to the bottle and incubated (Memmert ICO105 CO2 Incubator, adjusted use of 20% CO2) for 48 h at 37°C. Some of the solution and samples were tested for sterility by addition to the nutrient broth followed by incubation for 24 h at 37°C. Cell growth was examined every 24 h under an inverted microscope (Leica catalog number: DM IL LED) at 10×, 20×, and 40× magnification.\n\nAfter incubation for 48 h, the spent medium was removed and 25–30 mL of PBS was added. Then, 0.25% Trypsin Versene was added and incubated to dissociate the cells. After all cells had dissociated, fresh EM with 10% FBS and 3% PEG was added. Cell counting was performed with a hemocytometer (Fisher scientific catalog number: 22-600-107). Cell propagation was performed by repeating these three times to achieve stable growth. Cell passaging was performed to maintain cell growth for the antiviral assay.\n\nThe sample size was calculated using Equation 1, with an assumed {Z1 − (α/2)} at 1.96, with a significance of 0.05; Zβ at 1.645 by error limit of 5%; d at 3.62; Sa at 1.7; and Sb at 1.4.34 As the N value was rounded to 5, the control and test groups were each to consist of five samples.\n\nThe experiment was performed at a temperature of 4°C–5°C. The first step was the preparation of the QLCs at 0.05% (w/v) in the EM vehiculum by making the preparation of QLCs into a suspension through the addition of 2% Tween 40 p.g. Furthermore, antibiotics and antifungals were added and then filtered using a 0.20-μm filter. After filtration, the solution was ready to be used.\n\nFirst, the virus was diluted to 109 by mixing 0.3 mL of virus substance plus 2.7 mL of EM. Then, 0.3 mL of the mixture was taken and put in vials plus 2.7-mL EM, with slow shaking. A similar process was performed for up to the ninth dilution. The antivirus test was started by taking 0.1 mL of each virus dilution in a 96-well microplate. Each dilution was repeated in five microplate wells by the determination of the sample size. In each well, 0.3 mL of 0.05% QLCs and 0.1 mL of EM contained FBS 10% and polyethylene glycol 3% were used.\n\nThe control wells consisted of three groups: one group as a negative control, free from virus, a positive control, with a virus at 10−1 dilution, and a bioactive control with virus at dilution 10−1 with AEI 0.05.\n\nIn the last stage, all stocks were as follows; 0.1 mL of all test materials ranging from dilute viral substances to QLC and CK cultures and all EM, as well as 10% FBS and PEG, were taken and dropped to a 10 mL tube containing thioglycolate broth (Merck catalog number: 116761) to test for contamination. Then incubation was performed in a CO2 incubator at 37°C for 24 h. Then, an assessment of the antivirus activity was performed by starting with the examination of all nutrient broth in the tube. If the tube test was free from contamination, the assessment continued. Antivirus assessment consisted of assessment of the positive control and negative control first followed by the examination of each well for a cytopathogenic effect (CPE) using an inverted microscope (Leica catalog number: DM IL LED) at 10×, 20×, and 40× magnification.15 The examination technique for each microplate well and the CPE criteria are shown in Table 1.\n\nStatistical tests were performed using the probit analysis method for each virus dilution at which 50% antivirus activity was obtained for QLCs of 0.05% dilution, with a significance of α = 0.05. The statistical analysis was executed using the Statistical Package for the Social Sciences 24.0 software (IBM Corp., NY, USA).\n\n\nResults\n\nFrom a total of 5 kg BD leaves, approximately 5 g of flavonoid raffinates were obtained. For all raffinates, flavonoids were present as determined by both methods tested. These samples were known to contain flavonoids because of the change in color to green following the addition of a small amount of Zn into solutions with the raffinates and HCl. This was further tested by determining the Rf value of the chromatography thin-layer elution results and comparing it to that of the standard; the Rf value of QLCs (102 ppm, B) was equivalent to the Rf standard (100 ppm, C) as shown in Figure 1. The eluent mobile phase as a control appeared on the TLC marked as A. After elution, the substance spots were determined to be flavonoids after spraying with an ammonium solution revealed a red color (D).\n\nA. Control eluent, t mobile phase, B. Flavonoid. C. Standard quercetin. D. Flavonoid sprayed with ammonium solution giving a red color.\n\nThe analysis of the mobile phase fraction between water–methanol and the stationary phase in TLC is shown in Figure 2. Figure 2(A) shows that the adsorption–partition power against flavonoids was strong. TLC Figure 2(A); the left image shows QLCs and the right image shows the standards; Rf was 1.02. Figure 2(B), using a 3:7 phase fraction of mobile air–methanol shows the adsorption–partition power of QLCs (left) compared with the standard (right) moderate. The fraction of 3:7 was suitable for applications in HPLC (Rf 1). Figure 2(C), using the mobile air–methanol phase fraction of 2:8, shows that the adsorption–partition power of QLCs (left) compared with the standard (right) is weak and therefore unsuitable for applications in HPLC (Rf = 0.98). Spot QLCs are longer than Figures 2(A) and 2(B). Figure 2(D), the mobile fraction water–methanol at 1:9 shows that the adsorption–partition power of QLCs (left) compared to the standard (right) is very weak; the adsorbed partition spot becomes longer and is not suitable for applications in HPLC (Rf = 0.986).\n\nEach TLC strip contained two compounds: (left) samples and (right) standard of quercetin p.a.\n\nThe results of HPLC RT in Figure 3 show the peaks of the analyte (QLCs) in magenta and the standard in green color (Figure 3). The peak RTs of the two chromatograms were similar. The tolerance range between the retention times of analytes and the RT was estimated to be <5 min.\n\nThe IR spectrum of the standard and QLCs are shown in red and blue, respectively, in Figure 4. It is generally observed that the standard spectral intensity (%T) is greater in QLCs ranging from wavenumbers of 4000 cm−1 to 400 cm−1. However, there is still a higher intensity of QLCs than the standard, especially for wavenumbers of 2925 cm−1 to 2853 cm−1. However, in the fingerprint region, at wave numbers of 1200 cm−1 to 700 cm−1, there is no high spectral intensity. In general, Figure 4 shows that there are some similarities in the infrared spectra of the standard and the QLCs. The element water always appears at wavenumbers from 3600 cm−1 to 3000 cm−1, and this shows that the two compounds are polar and soluble in organic solvents. The specificity of the QLCs IR spectrum compared to the standard shows that there are peaks of the spectrum in the region before the fingerprint region and in the fingerprint region, although it has a %T value not too high. The IR spectra peaks for the analytes and standard that indicate similar compounds were the regions of 3420.40 cm−1 and 3399.45 cm−1 for the OH− ion; 1655.34 cm−1 and 1669.98 cm−1 assigned to the C=C ring, or 2- or 3-band stretching vibrations. The peaks at 1655.34 cm−1 and 1614.80 cm−1 for the analyte and 1665.98 cm−1 and 1613.65 cm−1 for the standard were assigned to the C=O bond. The other peak of 1515.42 cm−1 for the analyte and 1513.84 cm−1 for the standard were assigned as a C=C stretching. The peaks at 1458.39 cm−1 and 1378.66 cm−1 for the analyte and 1462.66 cm−1 and 1379.48 cm−1 for the standard were assigned to the –O–H bending of aromatic compounds. The peaks at 1316.49 cm−1 and 1246.52 cm−1 for the analyte and 1353.07 cm−1, 1319.20 cm−1, and 1244.80 cm−1 for the standard were assigned to an aromatic phenol ring. Other peaks, at 881.85 cm−1 and 808.47 cm−1 in the analyte and 881.90 cm−1 and 807.94 cm−1 in the standard, were assigned to a phenol –C=C– bond. The fingerprint area of analytes at 600.41 cm−1 and of the standard at 600.89 cm−1 indicated the same specific functional compounds of an aromatic or phenolic –COO– group.\n\nThe analysis of 1H NMR and 13C NMR is presented in Figure 5(A) and 5(B), respectively; in each part, the upper part shows the QLCs and the lower part the standard. From Figure 5(A), the proton spectrum of QLCs can be identified, namely the chemical shift (δ) at 4.800 ppm is CD3OD, while the chemical shift (δ) CD3OD of the standard quercetin spectrum is 4.870 ppm. Figure 5(B) shows the chemical shift (δ) position of the CD3OD in 13C NMR of QLCs and quercetin standards occurred at 47.665 ppm.\n\nAnalysis of BD plant QLCs and identification was performed based on the retention time for quercetin standards; five bioactive were identified as positively or negatively ionized molecular fractions (m/z). However, not all were identical when further traced to the amount of carbon, hydrogen and oxygen atoms bound to the aromatic rings of polyphenols. The results of the m/z (+) molecular mass-based analysis showed that QLCs have polyphenol elements similar to genistein, whereas m/z (−) results were similar to bioactive 5,7,8,3′,4′-pentamethoxyflavonone, 7-hydroxy-1-methoxy-2-methoxyxanthone, and pelargonidin 3-glucoside. Table 2 shows the molecular mass of the five bioactive compounds. Of the four bioactive compounds outside the standards, only one of had similarities with one of the standard mass molecular fragments, namely hydroxy-1-methoxy-2-methoxyxanthone. Further similarities between bioactive and hydroxy-1-methoxy-2-methoxyxanthone are shown in Figure 6.\n\nResults of the in vitro test of QLCs against NDV, after incubation for 24 h showed the nutrient broth in the control tube, was free from contamination. The observations at serial virus dilutions of 10−1 to 10−9 compared with the positive controls and negative controls are shown in Table 3 and Figure 7. Probit analysis of the 50% endpoint with a value of 10-2.314 showed that diluting the virulence of the NDVs was able to reduce its ability by half by observing CPE findings in CK culture (Figure 6, p<0.05).\n\n\nDiscussion\n\nThe separation of bioactive components to obtain QLCs from the crude methanol extract of BD leaves is easy in principle, but the yield is very small. For example, 5 kg of BD leaves yields approximately 250 mg of QLCs (0.005%). The low yield of QLCs obtained indicates that sample preparation must be carefully performed and indicates a potential loss of analytes during the separation and refining processes. One of the precautionary steps in the framework of laboratory work processes related to minimizing the loss of analytes is the identification of medicinal plants performed by an institution/authority. BD plants contain different active ingredients, and one of the areas where plants have the highest active ingredient content is the sampling location for this study (Muara Enim District, south of Sumatera, Indonesia).\n\nThe quality of bioactive content is different between areas owing to differences in nutrients, solar radiation received, and air quality. Enhancing these factors results in higher bioactive content in mistletoe. In Figure 1(B), the quality of flavonoids containing QLCs has Rf (1.04 cm), equivalent to quercetin (point C, 1.64 cm); the Rf is only 0.60 cm different. Spraying with ammonium produces a red color [Figure 1(D)]. These findings suggest that the separation of flavonoids from the crude extract of BD leaves has occurred correctly.\n\nThese results show that water–methanol fractions of 2:8 and also 1:9 was not satisfactory in separating the QLCs from other impurities. Thus, a fraction of 3:7 was selected as optimal. The most difficult part to separate is the components that contain halide elements that bind to non-halide elements and are toxic. To overcome this, it must be ensured that when withdrawing flavonoid elements, no elements are found that can cause organ damage through toxic elements in the extract. This has been confirmed for BD plants in previous studies.15,35\n\nBioactive QLCs appear in the chromatogram at RT of 12.376 min. The peak in the standard chromatogram occurred at RT of 12.736 min. Thus, QLCs will be found in retentate collected between 12.00 min to 13.00 min. Figure 3 shows that between the RTs of 12.00 min to 13.00 min, there are no impurities in the chromatogram peaks, so it is believed that those compounds collected in this RT range are QLCs. In this study, monitoring the peak appearance of the QLC chromatogram was carried out using three sample injections and one standard technique. The purpose of the technique was to monitor possible shifts in retention time for analytes. This was done considering that the peak area of standard natural ingredients such as quercetin resulting from HPLC analysis generally has more than one peak area. This was necessary because of the phenomenon of frequent shifting in the appearance of analyte and standard chromatograms when using HPLC. If the shift in the RT chromatogram lasts between 2–3 minutes, this can be tolerated. However, if it is >5 min, the column must be cleaned with the mobile phase so that the stationary column retains a high affinity for binding QLCs. From the standard chromatogram in Figure 3, the green graphic line shows that at RT 10.036 minutes other elements were found besides the main element (polyphenols), it is suspected that these elements were 2-hydroxy groups facing each other in the ortho molecular position of the aromatic ring. This structure is not found in QLCs, but if the analytes remain in the mobile phase solvent too long, then it is possible that the hydroxyl bond will break causing a peak to appear. To avoid the instability of the analytes, the time range for dissolving analytes in the eluent mobile phase is limited to 8 h.\n\nFT-IR analysis is intended to identify the similarity of functional groups through the fingerprint region, as shown by comparing the QLCs (blue spectrum) and quercetin (red spectrum) between 600 cm−1 and 400 cm−1 (Figure 4). However, QLC wave numbers in non-specific areas, namely between wave numbers 1800 cm−1 to 600 cm−1, can also be used as a study of the bioactive characteristics to be investigated. There are two things about FT-IR analysis, there was an IR spectrum with similar wavenumbers between QLCs and standards, but there was also an IR spectrum at certain wavenumbers that do not match QLCs with standards. The mismatch of the IR spectral between QLCs and standards was well understood considering that bioactive withdrawals in natural plant preparations were difficult to draw the purity of active ingredients from disruptive elements. The mismatch of the IR spectrum reaches up to 40% and this was often found in studies using plants as objects of study.36,37\n\nAnalysis of proton (1H) and carbon (13C) NMR of standard and analytes was performed using a 99.9% (w/v; 0.36 M) solution standard of quercetin and analytes dissolved in methanol-d4 were acquired using the JEOL JNM ECS-400 NMR spectrometer (Figure 5). This spectrum reveals three types of proton and carbon signals as follows: aromatic protons carbon and hydroxy protons carbon of the analyte and standard, and solvent proton carbon. The main structure of a QLC is a benzopyrone with three hydroxyl group substitutions (positions 3, 5, and 7) and at proton (1H NMR) positions of H-6 δ 6.198 ppm and H-8 δ 6.401 ppm. Other signals for the 2′,5′, and 6′ B ring of the quercetin structure were protonated at δ 6.902 ppm, δ 7.648 ppm, and δ 7. 751 ppm. The carbon NMR (13C NMR) 8, 10 of QLCs were present on the A ring of quercetin at δ 93.059 ppm and the AC ring of quercetin at δ 103.175 ppm. Other atoms of QLCs were present on the C ring of quercetin as follows: 2C δ 147.415 ppm, 3C δ 135.879 ppm, 4C δ 175.972 ppm, and 7C δ 164.215 ppm on the A ring of quercetin, and 9C δ 156.883 ppm on the AC ring of quercetin.\n\nThe results of the RT analysis of QLC molecules show that the similarity of RT to standards cannot be used as the main criterion. The combination of observations of the number of atoms, retention time, and the similarity of mass is required. In Table 2, it is shown that the mass (Da) of 5,7,8,3′,4′-pentamethoxyflavonone, and pelargonidin 3-glucoside is between 372–375 Da and 468–470 Da. The molecule in that period is not equivalent to the standard, which is between 302–303 Da. Thus, the two bioactive compounds are not very similar to QLCs. In terms of the molecular formula of the two bioactives, namely C20H22O7 for 5,7,8,3′,4′-pentamethoxyflavonone and C21H21ClO10 for pelargonidin 3-glucoside, they are not similar to the quercetin standard (C15H10O7). In contrast, genistein (C21H20O10) has a molecular mass of 432–434 Da, not very similar to the standard and is not included in the components forming QLCs. Thus, the compound that bears a resemblance to quercetin is 7-hydroxy-1-methoxy-2-methoxyxanthone and has a molecular structure of C15H10O6 as shown in Figure 6, at Electrospray Ionization-Mass Spectrometry (ESI-MS) (m/z) 431.10002, not too far from quercetin i.e., ESI-MS (m/z) 447.09509. Molecular ion fractions also found in (m/z) QLCs ESI-MS (−) 255.03051 show their proximity to molecular ion fractions (m/z) of standard ESI-MS (−) at 271.02536. The difference in ESI-MS (−) 15.99479 between the two can be understood by possible binding with other elements. Another molecular ion fraction is ESI-MS (m/z) 899.17656 for QLCs compared to ESI-MS (m/z) standard 185.19789 for standards, also bearing similarities.38\n\nThe results of in vitro studies show that the administration of a suspension of 0.05% QLCs can inhibit the NDV growth starting from 10−2 dilution, as shown in Table 2. Thus, the more diluted the virus, the more potent the QLCs suspension. Using probit analysis, the virulence ability of NDV following QLC administration of up to 25% will occur at dilution 10-2.937. The ability to reduce the virulence of NDV to zero by administration of 0.05% of QLCs was found for a dilution of 4.462. When considering the active structure and quercetin analogy, hydroxyl elements that are bound to a single aromatic element such as phenol, and located opposite each other to the ortho position have the ability to kill the NDV. It is known that the arrangement of the base pair (bp) of the NDV virus (rNDVs) is as follows: 5′AGCTTTGTTTAAACTTAGAAAAAATACGGGTAGAAGGCCACCATGGGCT130 GCCTGGGCAAC3′.39 The bp rNDVs have the ability to infect eukaryote aerobic cells, so they can easily attack the upper respiratory system and then infect the nerve cells of the brain.40 When associated with the molecular structure of QLCs, the hydroxy groups will bind to the bp part in the adenine guanine and tyrosine regions. The xanthone group contained in QLCs will also bind to adenine–quinine–tyrosine, thus inhibiting the process of viral replication in host eukaryotic cells without killing host cells.41 In such circumstances, the virus will gradually die itself. Xanthone derivatives will also bind to other bp such as cytosine with the effect of disrupting viral replication without causing death in host cells. Hydroxyl bonds and xanthone derivatives have a high affinity for base pairs that require high energy such as viral replication activities.42 The binding affinity will not target the base pairs of the host cell considering that the energy strength of viral replication is higher than the physiological activities of the host cell. The virucidal activity of QLCs is also influenced by the lipophilic nature of the BD extract compounds; they can easily penetrate the surface of the lipid bilayer of host eukaryotic cells. Thus, the QLC components kill the virus more quickly; this phenomenon is shown in Figures 7(B) and 7(C). The ability to utilize host cell organelles in NDVs is very high and this is needed for viral replication.43 In such circumstances, many host cells will die as seen in Figure 7(E) for the positive control and can be compared to the negative control 7(D). Replication behavior can be stopped with 0.05% AEI, and it can be seen in Figure 7(F) that CK cells are still growing, although are not very healthy with blackish elements in the medium. The black appearance of the medium in Figure 7(F) is clearly distinguished from control cells where the medium is dark red [Figure 7(A)].\n\nThe phenomenon of NDV viricide in 0.05% QLC suspension is thought to be suitable for use against other types of aerobic viruses such as foot and mouth diseases in cattle or lymph skin diseases in cattle, buffalo, sheep and goats. It is possible that it can be used against viral infections in humans.44 The virucidal potential of QLCs has good prospects, although an increase above a concentration of 0.05% does not necessarily result in higher virucidal power. This is due to the influence of the host’s base pair, which is at risk of disrupting the host cell metabolic cycle resulting in the death of the host cell. There are distinct advantages when using QLCs as a virucidal bioactive, namely the natural properties of these QLCs make them safe for the host body and do not carry the same risks as residues of veterinary drugs.45\n\n\nConclusions\n\nThe bioactivity of QLCs arises from the phenolic elements with the addition of hydroxyl groups and the aromatic structure of xanthone. These two elements can interact with viral rRNA base pairs. Thus, QLCs extracted from BD leaves are capable of exerting virucidal power against NDVs to a concentration of 0.05% (p<0.05). The viricidal power of 50 endpoints for QLCs against NDV was found with a dilution of 10−2.314. The weakening of NDV infection to 25% after administration of 0.05% QLCs occurred with a dilution of 10−2.937. The NDVs were eliminated completely after administration of 0.05% QLCs at a virus substances dilution of 10−4.462. The virucidal power does not interfere with the host cell’s metabolic processes; the host cell continues to grow and develop. Thus, bioactive QLCs are potential virucidal active compounds for both humans and animals. We hope that in the future they may be successfully developed into safe veterinary drug compounds.", "appendix": "Data availability\n\nFigshare: In vitro analysis of quercetin-like compounds from Mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease. https://doi.org/10.6084/m9.figshare.22587166. 46\n\nThis project contains the following underlying data:\n\n• In Vitro Test-Edit-1.spv (Output of probit analysis test groups)\n\n• Data of In Vitro Test-Edit-1.sav (Probit analysis data by cytopathogenic effect observation in microtiter plate, groups test vs. groups controls positive & control negative)\n\n• FT-IR Edit-1.pdf (Overlay the specific spectrum of Quercetin-Like Compounds vs Quercetin in wavelength number 400 cm-1 to 400 cm-1)\n\n• Certificate English editing.pdf (Certificate from ENAGO Corp., for editing grammar and sentences).\n\n• LC-ESI-MS.pdf (Analysis of Quercetin standard using LC-ESI-MS)\n\n• LC-ESI-MS.pdf (Analysis of Analytes as Quercetin-Like Compounds using LC-ESI-MS)\n\n• Data set in vitro test. pdf (An observed of cytopathogenic effect on the microtiter plates growth up well of virus NCD in chick kidney cells and add the analytes as Quercetin-Like Compounds)\n\n• Data statistic.pdf ((Dose-response as cytopathogenic effect of cells culture after add the dose dilution virus 1/10 to 1/100.000)\n\n• Nuclear Magnetic Resonance carbon of Analytes.pdf (Carbon NMR spectrum of analytes as an extract benalu duku)\n\n• Nuclear Magnetic Resonance proton of Analytes.pdf (Proton NMR spectrum of analytes as an extract benalu duku)\n\n• Nuclear Magnetic Resonance carbon of Standard Quercetin.pdf (Carbon NMR spectrum of standard as a quercetin)\n\n• Magnetic Resonance proton of Standard quercetin.pdf (Proton NMR spectrum of standard as a quercetin)\n\n• Spectrum FT-IR standard Quercetin and raw material Quercetin-Like Compounds.pdf (Spectrum Quercetin and Quercetin-Like Compounds at 4000 cm-1 to 450 cm-1)\n\n• Overlay peak area of HPLC.pdf (peak area of Quercetin vs. Quercetin-Like Compounds).\n\n• Screening flavonoid via Thin Layer Chromatograph.pdf (available of Quercetin-Like Compounds in flavonol of extract benalu duku.\n\n• In vitro test virus of NCD in chick kidney cell vs Quercetin-Like Compounds. Pdf (an analysis of the cytopathogenic effect of the virus after adding the Quercetin-Like Compounds).\n\n• Output analysis data statistic.pdf (Probit analysis: NSPE of the total with dose)\n\n• QTOF Water.jpg (Instrumentation for observing the LC-ESI MS)\n\n• Standard-Prof Laz carbon 1-2.jdf.pdf (Overlay standard carbon NMR)\n\n• Figure 7-A-FS.jpg (In vitro test of 0.05% quercetin-like compounds against live Newcastle disease virus at a dilution of 10-5 in chick kidney culture cells on 96-wells microplate. No cytopathogenic effect on culture cells was observed on magnified 40x by inverted microscope).\n\n• Figure 7-B-FS.jpg (In vitro test of 0.05% quercetin-like compounds against live Newcastle disease virus at a dilution of 10-4 in chick kidney culture cell on 96-wells microplate. Cytopathogenic effects were starting to appear on culture cells using an observed magnified 40x inverted microscope).\n\n• Figure 7-original 600dpi (2).jpg (Observes well of microplate at test in vitro)\n\n• Standard-Prof LAZ-31 Jan 2023_carbon1-2jdf.pdf (Analysis carbon NMR standard).\n\n• Figure 7-C-FS.jpg (In vitro test of 0.05% quercetin-like compounds against live Newcastle disease virus at a dilution of 10-4 in chick kidney culture cell on 96-wells microplate. Cytopathogenic effects in cell culture were very clear in the spaces between cells that were still alive observed by an inverted microscope on magnified 40x).\n\n• Figure 7-D-FS.jpg (Negative control of chicken kidney cell culture in Eagle’s medium grown on 96-wells microplate. Observed using 40x magnified of inverted microscope).\n\n• STANDAR-PROF LAZ-31_proton 1-3.jdf.pdf (An analysis proton NMR of Quercetin standard).\n\n• Figure 7-E-FS.jpg (Positive control of chick kidney cell culture containing of live Newcastle disease virus at a dilution of 10-1)\n\n• Negative control.jpg (Control negative in vitro test)\n\n• STANDARD-PROF LAZ-31JAN2023_proton-1-3.jdf.pdf (Analysis proton NMR of quercetin).\n\n• STANDARD-PROF LAZ-31 JAN203_PROTON-1-3.JDF.PDF-PERB1.pdf (Analysis proton NMR of extract BD)\n\n• 13-June-1.jpg (Research activities)\n\n• 13-June-2.jpg (Research activities)\n\n• 13-June-3.jpg (Research activities)\n\n• 13-June-4.jpg (Research activities)\n\n• 21-June-1.jpg (Research activities)\n\n• 21-June-2.jpg (Research activities)\n\n• 21-June-13.jpg (Research activities)\n\n• 15-July-1.jpg (Research activities)\n\n• 15-July-2.jpg (Research activities)\n\n• 15-July-3.jpg (Research activities)\n\n• 15-July-5.jpg (Research activities)\n\n• 15-July-6.jpg (Research activities)\n\n• 20-July-13.jpg (Research activities)\n\n• 1-August-1.jpg (Research activities)\n\n• 1-August-2.jpg (Research activities)\n\n• 1-August-3.jpg (Research activities)\n\n• 1-August-4.jpg (Research activities)\n\n• 1-August-5.jpg (Research activities)\n\n• 4-august-10.jpg (Research activities)\n\n• 5-August-1.jpg (Research activities)\n\n• 5-August-2.jpg (Research activities)\n\n• 5-August-3.jpg (Research activities)\n\n• 5-August-4.jpg (Research activities)\n\n• 9-August-8.jpg (Research activities)\n\n• 9-August-9.jpg (Research activities)\n\n• 9-August-10.jpg (Research activities)\n\n• 9-August-11.jpg (Research activities)\n\n• 9-August-12.jpg (Research activities)\n\n• 9-August-25.jpg (Research activities)\n\n• Figure 7 600dpi-edit New.jpg (In vitro test with 500 μm scale)\n\n• 10-August-3.jpg (Research activities)\n\n• 10-August-4.jpg (Research activities)\n\n• 10-August-5.jpg (Research activities)\n\n• 10-August-6.jpg (Research activities)\n\n• 30-August-1.jpg (Research activities)\n\n• 31-August-1.jpg (Research activities)\n\n• Oct-1.jpg (Research activities)\n\n• Sept-1.jpg (Research activities)\n\n• Kontrol positif & AEI 600dpi (1).jpg. (Positive control)\n\n• Kontrol positif & AEI 600dpi (1).jpg (Copy positive control)\n\n• In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra L. Miq as a potential antiviral agent for Newcastle disease.pdf (Webinar SATU)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe author is very grateful to the Rector of Universitas Airlangga and Head of Universities in Malaysia, namely UCSI, University of Malaya, University Sain Malaysia, who have supported these activities to collaborate under the SATU (South Asia and Taiwan University) Research Collaboration Program 2022. The author also thanks all veterinarians who have helped smooth this research from the beginning to the end of the work period.\n\n\nReferences\n\nLazuardi M, Bambang H: Dendrophthoe pentandra methanolic leaf extract increases progesterone levels in female rats. Univ. Med. 2014; 33(2): 100–108.\n\nChagas MSS, Behrens MD, Moragas-Tellis CJ, et al.: Flavonols and flavones as potential anti-inflammatory, antioxidant, and antibacterial compounds. Oxidative Med. Cell. Longev. 2022; 2022: 1–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLara-Guzman OJ, Tabares-Guevara JH, Leon-Varela YM, et al.: Proatherogenic macrophage activities are targeted by the flavonoid quercetin. J. Pharmacol. Exp. Ther. 2012; 343(2): 296–306. PubMed Abstract \| Publisher Full Text\n\nFerraz CR, Carvalho TT, Manchope MF, et al.: Therapeutic potential of flavonoids in pain and inflammation: Mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. Molecules. 2020; 25(3): 762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Petrillo A, Orrù G, Fais A, et al.: Quercetin and its derivates as antiviral potentials: A comprehensive review. Phytother. Res. 2022; 36(1): 266–278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazuardi M, Chien CH, Jie-Long H, et al.: In vivo analysis of extract of leaves of mistletoe as a Benalu Duku: Clinical chemical value associated with histopathology, liver, kidneys, and lethal dose determinate. Vet. Med. Int. 2022; 2022: 1–11. Publisher Full Text .\n\nSingh A, Singh DK, Kharwar RN, et al.: Fungal endophytes as efficient sources of plant-derived bioactive compounds and their prospective applications in natural product drug discovery: Insights, avenues, and challenges. Microorganisms. 2021; 9(1): 197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPajić M, Knežević S, Djurdjević B, et al.: Diagnosis of infectious laryngotracheitis outbreaks on layer hen and broiler breeder farms in Vojvodina, Serbia. Animals (Basel). 2022; 12(24): 3551. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh P, Arif Y, Bajguz A, et al.: The role of quercetin in plants. Plant Physiol. Biochem. 2021; 166(10): 10–19. Publisher Full Text\n\nPuro K, Sen A: Newcastle disease in backyard poultry rearing in the northeastern states of India: Challenges and control strategies. Front. Vet. Sci. 2022; 9: 799813. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRavishankar C, Ravindran R, John AA, et al.: Detection of Newcastle disease virus and assessment of associated relative risk in backyard and commercial poultry in Kerala, India. Vet. Med. Sci. 2022; 8(3): 1146–1156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMao Q, Ma S, Schrickel PL, et al.: Review detection of Newcastle disease virus. Front. Vet. Sci. 2022; 9: 936251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWodajo W, Mohammed N, Tora E, et al.: Sero-prevalence of Newcastle disease and associated risk factors in chickens at backyard chicken production Kindo Koisha, Wolaita zone, Southern Ethiopia. Front. Vet. Sci. 2023; 9: 1089931. Publisher Full Text\n\nAlexander DJ: Newcastle disease and other avian paramyxoviruses. Rev. Sci. Tech. 2000; 19(2): 443–462. Publisher Full Text\n\nRacioppi F, Martuzzi M, Matić S, et al.: Reaching the sustainable development goals through healthy environments: Are we on track? Eur. J. Pub. Health. 2020; 30(Suppl_1): i14–i18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazuardi M, Suharjono S, Chi-Hsien C, et al.: Toxicity test of flavonoid compounds from the leaves of Dendrophthoe pentandra (L.) Miq. using in vitro culture cell models. Vet. World. 2022; 15(12): 2896–2902. PubMed Abstract \| Publisher Full Text\n\nEdache EI, Uzairu A, Mamza PA, et al.: QSAR, homology modeling, and docking simulation on SARS-CoV-2 and pseudomonas aeruginosa inhibitors, ADMET, and molecular dynamic simulations to find a possible oral lead candidate. J. Genet. Eng. Biotechnol. 2022; 20(1): 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSathishkumar G, Gobinath C, Wilson A, et al.: Dendrophthoe falcata (L.f) Ettingsh (neem mistletoe): A potent bioresource to fabricate silver nanoparticles for anticancer effect against human breast cancer cells (MCF-7). Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014; 128(7): 285–290. Publisher Full Text\n\nRomero B, Susperregui J, Sahagún AM, et al.: Use of medicinal plants by veterinary practitioners in Spain: A cross-sectional survey. Front. Vet. Sci. 2022; 9: 1060738. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMochamad L, Hermanto B, Hestianah EP: Determination of progesterone compounds in the crude methanol extract of benalu duku leaves. Vet. World. 2019; 12(3): 358–366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArsène MMJ, Davares AKL, Viktorovna PI, et al.: The public health issue of antibiotic residues in food and feed: Causes, consequences, and potential solutions. Vet. World. 2022; 15(3): 662–671. Publisher Full Text\n\nNakamura K, Ito M, Nakamura T, et al.: Pathogenesis of Newcastle disease in vaccinated chickens: pathogenicity of isolated virus and vaccine effect on challenge of its virus. J. Vet. Med. Sci. 2014; 76(1): 31–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardiyanti R: The study of phytochemistry and bioactivity of flavonoid from Dendrophthoe pentandra (L. miq) Loranthaceae. [Doctor dissertation] Medan, North of Sumateran-Indonesia: The University of North Sumatera.2019.\n\nLazuardi M, Suharjomo S, Chi-Hsien C, et al.: Encapsulation of progesterone-like compounds in 10% liposome increases their concentration in rats administered an injectable dosage form of these compounds. Kafkas Univ. Vet. Fak. Derg. 2021; 28(1): 27–34.\n\nXiao Y, Fu F, Wei Y, et al.: Online extraction-DPPH-HPLC-DAD-QTOF-MS system for efficient screening and identification of antioxidants from Citrus aurantium L. var. amara (Rutaceae): Integrating sample preparation and antioxidants profiling. Antioxidants (Basel). 2022; 11(5): 1014. Publisher Full Text\n\nKokalj LM, Straus J, Tavčar BE, et al.: FT-IR-based method for rutin, quercetin and quercitrin quantification in different buckwheat (Fagopyrum) species. Sci. Rep. 2017; 7(1): 7226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoshi R, Sathasivam R, Jayapal PK, et al.: Comparative determination of phenolic compounds in Arabidopsis thaliana Leaf powder under distinct stress conditions using Fourier-transform infrared (FT-IR) and near-infrared (FT-NIR) spectroscopy. Plants (Basel). 2022; 11(7): 836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHssaini L, Razouk R, Bouslihim Y: Rapid prediction of fig phenolic acids and flavonoids using mid-infrared spectroscopy combined with partial least square regression. Front. Plant Sci. 2022; 13: 782159. Publisher Full Text\n\nLazuardi M, Hermanto B: LC ESI-MS and FT-IR analysis of Dendrophthoe pentandra L. Miq leaf methanolic extracts to identify compounds with progesterone-like effects. Pak. J. Nutr. 2016; 15(3): 274–282. Publisher Full Text\n\nMohammed HA, Almahmoud SA, El-Ghaly EM, et al.: Comparative anticancer potentials of taxifolin and quercetin methylated derivatives against HCT-116 cell lines: Effects of O-methylation on taxifolin and quercetin as preliminary natural leads. ACS Omega. 2022; 7(50): 46629–46639. Publisher Full Text\n\nEl-Nashar HAS, El-Labbad EM, Al-Azzawi MA, et al.: A new xanthone glycoside from Mangifera indica L.: Physicochemical properties and in vitro anti-skin aging activities. Molecules. 2022; 27(9): 2609. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOliva E, Fanti F, Palmieri S, et al.: Predictive multi experiment approach for the determination of conjugated phenolic compounds in vegetal matrices by means of LC-MS/MS. Molecules. 2022; 27(10): 3089. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdulhafiz F, Reduan MFH, Hisam AH, et al.: LC-TOF-MS/MS and GC-MS based phytochemical profiling and evaluation of wound healing activity of Oroxylum Indicum (L.) Kurz (Beka). Front. Pharmacol. 2022; 13: 1050453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHennion RM, Hill G: The preparation of chicken kidney cell cultures for virus propagation. Methods Mol. Biol. 2015; 1282: 57–62. Publisher Full Text\n\nBejoy J, Qian ES, Woodard LE: Tissue culture models of AKI: From tubule cells to human kidney organoids. J. Am. Soc. Nephrol. 2022; 33(3): 487–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazuardi M, Hestianah EP, Restiadi TI: Designing prototype rapid test device at qualitative performance to detect residue of tetracycline in chicken carcass. Vet. World. 2022; 15(4): 1058–1065. PubMed Abstract \| Publisher Full Text\n\nSkrypnik L, Feduraev P, Golovin A, et al.: Biotechnological potential of different organs of mistletoe (Viscum album L.) collected from various host tree species in an urban area. Plants (Basel). 2022; 11(20): 2686. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWulandari L, Retnaningtyas Y, Nuri LH: Analysis of flavonoid in medicinal plant extract using infrared spectroscopy and chemometrics. J. Anal. Methods Chem. 2016; 2016: 1–6. Publisher Full Text\n\nKokalj Ladan M, Straus J, Tavčar Benković E, et al.: FT-IR-based method for rutin, quercetin and quercitrin quantification in different buckwheat (Fagopyrum) species. Sci. Rep. 2017; 7(1): 7226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi ZH, Guo H, Xu WB, et al.: Rapid identification of flavonoid constituents directly from PTP1B inhibitive extract of raspberry (Rubus idaeus L.) leaves by HPLC-ESI-QTOF-MS-MS. J. Chromatogr. Sci. 2016; 54(5): 805–810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun Y, Zheng H, Yu S, et al.: Newcastle Disease Virus V Protein Degrades Mitochondrial Antiviral Signaling Protein To Inhibit Host Type I Interferon Production via E3 Ubiquitin Ligase RNF5. Virol. J. 2019; 93(18): e00322–e00319. Publisher Full Text\n\nHu Z, He X, Deng J, et al.: Current situation and future direction of Newcastle disease vaccines. Vet. Res. 2022; 53(1): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu Y, Scholle F, Kisthardt SC, et al.: Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E. Virology. 2022; 571: 21–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrüger N, Kronenberger T, Xie H, et al.: Discovery of polyphenolic natural products as SARS-CoV-2 Mpro inhibitors for COVID-19. Pharmaceuticals. 2023; 16(2): 190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChathuranga WAG, Hewawaduge C, Nethmini NAN, et al.: Efficacy of a novel multiepitope vaccine candidate against foot-and-mouth disease virus serotype O and A. Vaccines. 2022; 10(12): 2181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazuardi M, Khairat J, Lim V, et al.: In vitro analysis of quercetin-like compounds from Mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease. [Dataset]. figshare. Publisher Full Text" }	[ { "id": "214625", "date": "30 Oct 2023", "name": "Adel M. Abdelaziz", "expertise": [ "Reviewer Expertise Avian disease and medicine", "avian virology", "molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is clear and sound as in vitro study of the antiviral agent, but the evaluation of antiviral activity of any compound need field trial in vivo by challenge test with field virus strain on vaccinated and non-vaccinated bird treated with the compound. The effect of these plant compound is to enhance the immunity either humoral or cell mediated immunity, in either vaccinated and non-vaccinated bird.\nBy answering and addressing these observations, the research can be accepted. If they are not addressed, the research cannot be accepted:\nThe field virus\n\nChallenge virus need to assist its velogenicity by mean death time (MDT) or other known pathogenicity index as (IVPI OR ICPI).\n\nDo you have any data or another method about assessment of virulene of field virus strain?\n\nDo you have any reference about the pathogenicity assessment of field virus?\n\nDo you have any reference about (Modified field test)? If present please put it.\n\nDo you have any reference for the method of egg injection method? Or correct it.\nIn abstract\nRemove the word 'and in, \"...and in chicken kidney cell culture.\"\n\nCorrect the word 'virulency' in abstract to be, 'virulence'.\n\nCorrect the conclusion section in abstract as follows: \"...QLCs from flavonoids from the leaves of BD have invetro antiviral bioactivity against NDVs against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10501", "date": "07 Nov 2023", "name": "Prof. Lazuardi Mochamad", "role": "Author Response", "response": "Comment: 1. Challenge virus need to assist its velogenicity by mean death time (MDT) or other known pathogenicity index as (IVPI OR ICPI). Response: The data of the virulence test of the field virus had been described in Table 2 and Table 3 2.Do you have any data or another method about assessment of virulene of field virus strain? Response: This research did not screen the strain yet, but for vaccination and to challenge the live virus we used a homologous strain as an Indonesia PUSVETMA Strain of NDV at titer HA and HI assessed method. The new references at 36 was added to the list of references. 3. Do you have any reference for the pathogenicity assessment of field virus? Response: The new reference at 37 and 38 4. Do you have any reference about (Modified field test)? If present please put it. Response: This has been described in Suggest of this research parts. 5. Do you have any reference for the method of egg injection method? Or correct it. Response: Will be described in the Suggest of this research part and add the new reference at no. 49 and 50" } ] } ]	2	https://f1000research.com/articles/12-1214
https://f1000research.com/articles/12-1437/v1	06 Nov 23	{ "type": "Study Protocol", "title": "Development, implementation, and testing of LGBTQIA+ care curriculum for health science professionals: Research protocol.", "authors": [ "Mamatha Shivananda Pai", "Renjulal Yesodharan", "Vikram Palimar", "Latha Thimmappa", "Bhavana B. Bhat", "Nirmal Krishnan M.", "Deeksha Shetty", "Bontha V. Babu", "Mamatha Shivananda Pai", "Vikram Palimar", "Latha Thimmappa", "Bhavana B. Bhat", "Nirmal Krishnan M.", "Deeksha Shetty", "Bontha V. Babu" ], "abstract": "Lesbian, Gay, Bisexual, Transgender, Queer, Intersex and Asexual (LGBTQIA+) people struggle to identify a healthcare service that understands their problems and needs. Additionally, healthcare professionals also find it difficult to care for LGBTQIA+ as very little is studied or heard about management. The article presents a protocol for a pilot study aimed at the development of an LGBTQIA+ care curriculum for health science professionals. The study includes Phase I: The development of a curriculum based on a literature review and focus group discussion among LGBTQIA+ individuals, and Phase II: Pilot testing of LGBTQIA+ care curriculum. The study outcome will reflect the improvement in the knowledge of healthcare professionals on LGBTQIA+ care.", "keywords": [ "Curriculum", "Faculty", "Health Sciences", "Health professional", "India", "LGBTQIA+", "Research Protocol", "Students", "SDG 3", "SDG 10" ], "content": "Background\n\nThe Lesbian, Gay, Bisexual, Transgender, Queer, Intersex, and Asexual (LGBTQIA+), are a varied group of people with different gender identities, sexual orientations, and reproductive development. All members of the LGBTQIA+ community have different healthcare-related problems and requirements, even though they are sometimes grouped as a coalition. However, they share the stigma and discrimination that have hindered them from accessing quality healthcare in several ways (Agarwal & Thiyam, 2022; MacKenzie et al., 2020). Human health and social systems are interrelated. According to a Yang (2021)’s study, LGBTQIA+ individuals confront social challenges such as stigma, homophobia, discrimination, coming out, insufficient social support systems, and a lack of LGBTQIA+-friendly medical resources that not only put them in danger for physical and mental harm but also put their health at risk. LGBTQIA+ individuals face discrimination, violence, and bullying as they come to terms with their identity. LGBTQIA+ individuals are more vulnerable to mental health problems including anxiety and depression as a result of this type of minority stress (Yang, 2021).\n\nIndividuals can self-identify in different ways, including gender identity and sexual orientation. For the remainder of the text, we will refer to this spectrum collectively as the LGBTQIA+ community. The terms “L” for lesbian and “G” for gay refer to people who are attracted to people of the same gender; “B” for bisexual means they are attracted to people of both genders; and “T” for transgender means they identify as a gender other than the one they were given at birth. The ‘+’ recognizes and includes additional identities and orientations not covered by the initials and refers to the community’s dynamic nature (Agarwal & Thiyam, 2022). A person’s sexual and emotional attraction to another person, any resulting behaviour, and/or social connection are all considered to be part of their sexual orientation. The strongly held, innate notion that a person has of being a boy, a man, or another member of the male, female, or alternate gender is considered their gender identity. The way society views homosexuality varies widely between countries and historical eras. Heterosexuality is now accepted as the standard across the globe. LGBTQIA+ individuals face stigma and stereotypes in many countries. Although the Delhi High Court decriminalized homosexuality in 2009, the Indian Supreme Court affirmed section 377 of the Indian Penal Code, which makes adult consenting same-sex contact a crime, on December 11, 2013 (Kar et al., 2018). The Supreme Court of India recognized LGBTQIA+ people as the third gender in April 2014, and any discrimination against them was viewed as a violation of their constitutional rights.\n\nDespite recent developments in the acceptance of LGBTQIA+ individuals, education on LGBTQIA+ health requirements for health professionals still lags far behind despite evidence showing a tremendous rise in LGBTQIA+ acceptance and the achievement of equality in many sectors. Multiple studies and reviews of health issues have shown a persistent gap in healthcare education, with no standard texts that include information concerning care for LGBTQIA+ individuals (Keuroghlian et al., 2017). LGBTQIA+ community members encounter several difficulties regarding sexual orientation and gender identity in a heteronormative culture. Additionally, they frequently experience prejudice and sexual assault. Further, their medical requirements are more likely to be overlooked or socially rejected due to their sexual orientation and gender identity, which can affect their medical rights and the medical care they receive. LGBTQIA+ individuals frequently struggle with coming out when dealing with medical professionals. Two things worry them; First, medical professionals’ ignorance, bias, and discrimination may impair their right to get medical care. Second, incomplete information disclosure may influence a disease’s diagnosis or possibly lead to a misdiagnosis. LGBTQIA+ individuals must carefully balance the risks of coming out with the benefit of having the right medical attention and support. These factors frequently lead to psychological pressure, which is harmful to both physical and mental health (Yang, 2021).\n\nThe stigmatized and discriminated populations must be addressed by healthcare practitioners. Understanding how medical students feel about homosexuality is crucial for improving the healthcare system. Patients who identify as LGBTQIA+ have encountered stigmatization, discrimination, and even refusal of care within the healthcare system (Kar et al., 2018). In 2017, the Joint United Nations Programme on HIV/AIDS UNAIDS report stated that LGBTQIA+ individuals made up 4.3% of the population in India who were at high risk of contracting AIDS (Kar et al., 2018). Due to the underrepresentation of such information in medical school curricula, clinicians may not be aware of or sensitive to the needs and issues faced by LGBTQIA+ patients when they encounter them (Sequeira et al., 2012; Magnus & Lundin, 2016).\n\nHealthcare professionals are frequently not trained in or sensitive to the requirements of LGBTQIA+ individuals’ health. Additionally, it might be challenging for professionals to talk about identity in general, especially when it comes to sexual orientation and gender identity. Medical education institutions can sometimes become the breeding ground for a heteronormative ideology that supports heterosexualism (Lundin, 2011; Magnus & Lundin, 2016). Heteronormativity refers to the belief that only two opposite and mutually complementary genders exist – or that gender and sexual variation simply does not exist in social institutions (Kannisto, 2019). Due to a lack of specialized expertise and/or heterosexist attitudes on the part of healthcare personnel, LGBTQIA+ people face barriers to accessing sufficient healthcare in this way. The heterosexist attitudes may result in inaccurate risk assessments for sexually transmitted infections (STIs), pregnancy, and the inadequate or incorrect use of screening equipment. These obstacles may negatively affect the treatment management and, ultimately, the health of these people (Wahlen et al., 2020).\n\nThe LGBTQIA+ community is substantially more likely to experience various risk factors for poor health than heterosexual people, such as being less likely to have health insurance, being more likely to be obese, smoking more regularly, and engaging in binge and heavy drinking, the population’s age warrants additional attention. In addition, compared to heterosexual women, lesbian and bisexual women may undergo fewer preventative screenings for colon, breast, and cervical cancer. This is partly due to fear of not receiving respectful healthcare. Healthcare professionals are at the forefront of this effort and have the chance to treat everyone with respect, regardless of their sexual orientation and gender identity. Lack of time, finances, education, and clinical experience are a few obstacles that can prohibit physicians from providing respectful medical care (Walker et al., 2016). Doctor-patient interaction is essential for enhancing people’s health (Parker & Bhugra, 2000). Patients may opt to keep their sexual orientation and gender identity private. In such situations, healthcare professionals must be vigilant and compassionate to deliver the best care (Grabovac et al., 2014).\n\nThere are no such policies or curricula for treating LGBTQIA+ patients in India’s healthcare sector. LGBTQIA+ people have common social tendencies and decisions that impact their behavior while seeking healthcare, preventative health measures, and illness risk (Kaufman et al., 2014). It is vital to make accessible, responsive, appropriate, and well-resourced healthcare services provided by knowledgeable and trained healthcare professionals to support a better patient experience. Higher education institutions and healthcare organizations have a significant role in developing curricula (Cui, 2023) that can be accessed by all groups, including those who identify as LGBTQIA+ (McCann & Brown, 2018). Health institutions need to make room for individuals from different sexual orientations and gender identities and communicate a sense of welcome (Hafford-Letchfield et al., 2017).\n\nTo better prepare medical students to treat these underserved areas and lessen healthcare inequities, medical teachers play a crucial role (Alhanachi et al., 2021; Chinchilla & Arcaya, 2017). Training medical students during their studies can help them feel more at ease when caring for these patients and give better treatment, an essential technique for improving understanding and attitudes about LGBTQIA+ persons among physicians (Wahlen et al., 2020). Inculcating positive LGBTQIA+ attitudes among medical professionals plays a great role in reducing homophobia and transphobia (Gegenfurtner, 2021). The scope of the proposed research is to develop a curriculum on the care of LGBTQIA+ and pilot test on faculty and students of health sciences in the form of workshops. For this purpose, a need assessment through a review of the literature and focus group discussion with LGBTQIA+ individuals will be done.\n\n\nResearch plan\n\nHow is an LGBTQIA+ curriculum effective on the knowledge of healthcare providers in caring for LGBTQIA+ individuals?\n\nPrimary research question\n\nWhat are the healthcare needs of LGBTQIA+ people?\n\nTo answer the primary research question, lead questions will be used to assess LGBTQIA+ individuals’ care needs, barriers to accessing care, and expectations from healthcare providers.\n\nSecondary research question\n\nWhat is the knowledge of health science students and faculty on LGBTQIA+ health needs?\n\nTo achieve this question, a structured knowledge-based questionnaire on LGBTQIA+ care will be administered to the health science students and faculty to assess their knowledge about LGBTQIA+ care.\n\nHypothesis/Assumptions\n\nIt is hypothesized that a curriculum on LGBTQIA+ care will significantly increase the knowledge of the healthcare professional in the care of LGBTQIA+ individuals.\n\nBased on the previously published literature related to the needs of LGBTQIA+; it is assumed that\n\na. LGBTQIA+ individuals experience stigma and discrimination.\n\nb. The present health science curriculum does not have a specific curriculum that deals with the management of LGBTQIA+ health problems.\n\nc. There is a lack of structured guidelines for managing the health conditions of LGBTQIA+ people.\n\nDesign\n\nThe study includes two phases: Phase I - development of the curriculum based on the literature and the need assessment through the focus group discussion among LGBTQIA+ individuals; and Phase II - A pilot testing of the curriculum for health science faculty and students with pre- and post-test assessment design. A conceptual framework of the research design is shown in Figure 1.\n\na. Literature review\n\nThe LGBTQIA+ curriculum for health sciences will be developed based on the detailed literature review and analysis of focus group discussion (FGD). The literature will be reviewed in detail through different sources to understand the needs of the LGBTQIA+ community. This includes academic research databases such as SCOPUS, Web of Science, PubMed, Science Direct, and CINAHL Complete. The grey literature search will also be initiated. The literature search is primarily conducted to find out the existing health care needs of the LGBTQIA+ community and it does not involve the steps of systematic reviews. The literature on the needs, problems, and expectations of LGBTQIA+ will be studied and used for developing the curriculum.\n\nb. Focus group discussion\n\nThe FGD will be conducted in a district government office in coordination with the health department of the local government. The Principal Investigator will moderate the FGD by following the standard methodology (Kitzinger, 1995). The data will be collected from the participants after obtaining administrative permission from the authorities. The participant information will be explained in the local language, and a signed informed consent will be taken from the participants for collecting the data and for audio recording. The sociodemographic data of the participants will be taken, and the participants will self-report their sexual orientation and gender identity. The lead questions prepared for the FGD will be used for conducting the discussion (Box 1). After receiving consent from the participants, a discussion will be initiated using the lead questions. Participants are encouraged to communicate and discuss their healthcare needs and expectations from the healthcare facility. Based on the participant’s response, probe questions will be asked till all the questions are answered. The FGD will be recorded using an audio recorder with the consent of the participants, and nonverbal communication during the discussion. It will be used as an adjunct while transcribing the discussion. A sociogram also will be drawn to record the interactions among the participants. The FGD session will be closed by the moderator after summarizing the important points to the participants. The team will thank the participants and compensate them for the quality time spent by the participants. The data gathered will be thematically analyzed (Braun & Clarke, 2021; Kyngäs et al., 2019), and will be used for developing the curriculum for health science students.\n\na. Describe your experience of visiting a hospital/clinic. (Probe-interaction with health professionals)\n\nb. What are your health needs that require medical attention?\n\nc. Explain the changes you expect in addressing your health needs.\n\nd. What are your expectations in addressing you?\n\ne. What are your expectations from the health care provider when you visit the hospital/clinic?\n\nf. How can a healthcare provider best assess your health needs?\n\ng. How can healthcare providers help you in the treatment of any illness?\n\nh. What are the challenges you face in seeking treatment for your health needs?\n\ni. Explain any other information you would like to bring to our attention.\n\nc. Development of LGBTQIA+ care curriculum\n\nBased on the literature review and FGD analysis, the authors will draft the LGBTQIA+ Care curriculum. The draft will be sent to experts for content validation, and the final curriculum will be developed based on the expert’s opinions. The developed curriculum for the care of LGBTQIA+ individuals will be used for Phase II.\n\nThe curriculum will be pilot-tested in the form of workshops for faculty and students of health sciences in the workplace. The faculty and students will be from nursing, medicine, psychology, and nurse educators. The workshop will be for one day. Knowledge will be assessed before and after the workshop using a questionnaire. The tentative topics covered in the workshop are, introduction to LGBTQIA+ care, inclusive communication, LGBTQIA+ community and health care- treatment management, mental health services for LGBTQIA+, research and evidence-based practice, interpersonal communication, ethical and legal issues in LGBTQIA+ health care. Feedback from the participants will be also taken for the modification of the curriculum. The curriculum will be finalized after the pilot study, and recommendations will be submitted to health sciences institutions, their regulatory bodies, and funding agencies.\n\nPhase I\n\nPhase I includes an FGD to assess the healthcare needs among self-identified LGBTQIA+ individuals. The result of the FGD and the literature review will help in preparing the curriculum.\n\nOne face-to-face FGD of 10-15 individuals who are a representative number of the self-reported LGBTQIA+ community will be recruited using purposive sampling. The details of the participants will be collected from the district authorities. They will be contacted to schedule the FGD. The sample size is kept under 10-15 to support the depth of FGD and subsequent analysis.\n\nInclusion criteria\n\n• The participants who self-report as LGBTQIA+ community and can speak in Kannada or English.\n\n• Individuals who are above the age of 18 years.\n\n• Individuals who are willing to participate in the focus group discussion.\n\nExclusion criteria\n\n• Any person who is not belonging to the LGBTQIA+ community.\n\n• Any individuals who are crossdressers.\n\n• Any member of the LGBTQIA+ community who has a diagnosed mental disorder listed under chapter F of ICD 10, except gender identity disorder.\n\nDrop-out criteria and withdrawal\n\nData collection from participants will be ceased when they withdraw their consent to participate, and the concerned data will be excluded during analysis.\n\nThe participants for phase II will be selected from the health sciences institutions offering medical, nursing, and other health science programs through their heads of the institution. Faculty and students will be recruited face to face to the study, and the curriculum will be pilot-tested both among the faculty and students through workshops. No dropouts are expected as the workshop is for one day.\n\nThe number of participants for this group will be based on the formula:\n\nwhere;\n\nα (two-tailed) = % Threshold probability for rejecting the null hypothesis. Type I error rate.\n\nβ = % Probability of failing to reject the null hypothesis under the alternative hypothesis. Type II error rate.\n\nE = Effect size\n\nSΔ = Standard Deviation of the change in the outcome\n\nZα = Standard normal deviate for α = 1.9600\n\nZβ = Standard normal deviate for β = 0.8416\n\nB = (Zα + Zβ)2 = 7.8489\n\nC = (E/SΔ)2 = 0.2844\n\nN = B/C =27.5937\n\nThe N thus calculated is rounded up to 30 participants from students and faculty groups.\n\nStudents from medical (n = 10), nursing (n = 10), clinical psychology (n = 5), and senior nurses from the teaching hospital (n = 5) will be trained in the ‘student workshop’ whereas faculty from medicine (n = 10), nursing (n = 10) and psychology (n = 5) and middle-level nurse managers or educators (n = 5) will be trained in the ‘faculty workshop’. Undergraduate students and faculty are excluded from the ‘student workshop’, and similarly, students are excluded from the ‘faculty workshop’.\n\nThe outcome variables will be:\n\nPhase I:\n\n• Needs of the LGBTQIA+ individuals (analyzed) from FGD\n\n• Curriculum on LGBTQIA+ for the health professionals\n\nPhase II:\n\n• Knowledge of health professionals on the care of LGBTQIA+ individuals.\n\nPrimary outcome\n\nThe healthcare needs of LGBTQIA+ individuals will be assessed, and the data gathered will be used in developing the curriculum for health science students.\n\nSecondary outcome\n\nThe curriculum developed using the data gathered by FGD, literature review, and experts’ inputs will positively influence how LGBTQIA+ individuals are getting cared for by healthcare providers.\n\nPlan for data analysis\n\nPhase I\n\nThe FGD will be analysed by thematic analysis (Braun & Clarke, 2021; Renjith et al., 2021). The focus of the FGD analysis is not to identify the individual contributions to the discussion but to present the spectrum of opinions of the entire group (Van Eeuwijk & Angehrn, 2017). The data will be divided into simpler text units for coding and the coding will be done manually. Units of meaningful text corresponding to similar codes will be grouped and categorized systematically by the authors. Any differences in the process of coding and categorizing will be resolved by discussion among the authors. Consensus will be achieved during these face-to-face discussions. The codes will be categorized into inductive and deductive and inductive codes will be content-driven and raised by participants, whereas deductive codes originated from the discussion guide and will then be verified with data (Hennink et al., 2019).\n\nPhase II: Descriptive statistics like frequency and percentage will be used for the data analysis of the workshop participants. The knowledge data will be analysed using a paired t-test.\n\nDissemination\n\nResults will be disseminated via presentations at appropriate scientific conferences and meetings of professional bodies. The study will also be published in peer-reviewed journals, professional and institutional repositories, etc. The results will be discussed with governmental bodies and other stakeholders for broader implementation.\n\nStatus of the study\n\nThe study team completed the draft curriculum and is currently in the process of pilot testing the LGBTQIA+ care curriculum (phase II). The study is expected to be completed by December 2024, and the results will be published by 2024 and 2025. This protocol will help in reducing unnecessary duplication of effort and the costs of future studies.\n\n\nDiscussion\n\nThe previous study findings show that LGBTQIA+ individuals face more difficulties accessing care because of their sexual orientation and gender identities. The study found that young adult lesbians had a harder time getting access to care than young adult homosexual males. This finding is consistent with earlier research that identified differences in the healthcare experiences of sexual minority individuals. Additionally, due to negative experiences associated with their sexual orientation and identity, gay males were more prone than lesbians to delay care. Young men may be more forthcoming about their sexual orientation and identity in healthcare, which may increase the potential for negative experiences, whereas young women may be more reticent to disclose their sexual orientation and identity to providers and as a result feel limited in their ability to access affirming care (Macapagal et al., 2016).\n\nContrary to earlier findings, few individuals claimed to have encountered LGBTQIA+-related discrimination in medical settings. Furthermore, the majority stated that telling their provider about their gender identity and sexual orientation had a neutral to a positive impact on their care (Mosack et al., 2013). The findings from previous studies suggested that changes in the healthcare system will promote inclusive care. Studies have shown that reluctance to talk about sexual orientation and gender identity was brought on by ignorance of the medical requirements for LGBTQIA+ patients (LaVaccare et al., 2018). According to a previous research study, students who had more contact with LGBTQIA+ patients were more likely to ask about a patient’s sexual orientation and gender identity and check for children in the patient’s family. The disclosure of this information during patient interactions may be improved by early intervention by educators who teach students appropriate questions to ask during the history-taking process (Sanchez et al., 2006). The current study will explore the healthcare needs of LGBTQIA+ and will result in education programs and initiatives that can improve knowledge about LGBTQIA+ individuals and will provide practical techniques that can easily be included in the health science curriculum which will help in reducing the disparities. The curriculum will improve the healthcare professionals’ knowledge about sexual orientation, gender identity, sexual behaviour, and sex anatomy comfort of LGBTQIA+ patients. It will also enhance the self-confidence and comfort of the healthcare professionals to treat LGBTQIA+ people. The curriculum will summarize the healthcare needs, barriers, and expectations based on FGD and primary care recommendations for LGBTQIA+ patients. This study will support the necessity for a curricular framework to reduce unconscious bias among students of healthcare professions toward LGBTQIA+ patients.\n\n\nConclusion\n\nThe study will address the critical gap in the medical curriculum about LGBTQIA+ care. The curriculum will enhance the skills and knowledge of the healthcare providers about the needs and expectations of LGBTQIA+ individuals. It will promote healthcare professionals' positive attitudes toward LGBTQIA+ patients and improve comfort working with LGBTQIA+ patients. The curriculum will guide researchers and educators looking to reduce prejudice against LGBTQIA+ patients in healthcare professionals, as well as a framework for teaching students to recognize and overcome their own biases. Educational Strategies that reduce bias in healthcare providers are essential steps to improving LGBTQIA+ communities' access to treatment and reducing health inequalities.\n\nFigure 2 depicts the activities and publications that will be carried out throughout the project. The project will endure for two years.\n\nThe Institutional Ethics Committee of Kasturba Medical College and Kasturba Hospital reviewed and approved the proposal on 11th May, 2022 (IEC1-138/2022). The protocol has been registered to the Clinical Trial Registry-India. Permission has been obtained from the local government authorities concerned, and written informed consent has been taken from the participants in the study. The data relating to the participants will be kept confidential and used, anonymously for this study only. Codes will be used for each participant.\n\nThe curriculum developed using the data given by the participants will positively influence how LGBTQIA+ individuals are being cared for by healthcare providers. A timely contribution to discussions concerning the function of professional educational interventions is made by this project, which evaluates the impact of educational curricula and training for healthcare students and professionals on LGBTQIA+ healthcare issues. A policy document will be made at the end of the study by highlighting the study’s implications and will be disseminated among the ministries and other regulatory bodies of health and education.", "appendix": "Data availability\n\nNo underlying data are associated with this article.\n\n\nReferences\n\nAgarwal A, Thiyam A: Healthcare, culture & curriculum: Addressing the need for LGBT+ inclusive medical education in India. Lancet Reg. Health Southeast Asia. 2022; 8: 100085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlhanachi S, de Meijer LAL , Severiens SE: Improving culturally responsive teaching through professional learning communities: A qualitative study in Dutch pre-vocational schools. Int. J. Educ. Res. 2021; 105: 101698. Publisher Full Text\n\nBraun V, Clarke V: To saturate or not to saturate? Questioning data saturation as a useful concept for thematic analysis and sample-size rationales. Qual. Res. Sport Exerc. Health. 2021; 13(2): 201–216. Publisher Full Text\n\nChinchilla M, Arcaya MC: Using Health Impact Assessment as an interdisciplinary teaching tool. Int. J. Environ. Res. Public Health. 2017; 14(7): 744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui L: Out queer teachers as role models for queer students? Implications drawn from closeted gay academics’ experiences in China. Int. J. Educ. Res. 2023; 117: 102137. Publisher Full Text\n\nGegenfurtner A: Pre-service teachers’ attitudes toward transgender students: Associations with social contact, religiosity, political preference, sexual orientation, and teacher gender. Int. J. Educ. Res. 2021; 110: 101887. Publisher Full Text\n\nGrabovac I, Abramović M, Komlenović G, et al.: Attitudes towards and knowledge about homosexuality among medical students in Zagreb. Coll. Antropol. 2014; 38(1): 39–45. PubMed Abstract\n\nHafford-Letchfield T, Pezzella A, Cole L, et al.: Transgender students in post-compulsory education: A systematic review. Int. J. Educ. Res. 2017; 86: 1–12. Publisher Full Text\n\nHennink MM, Kaiser BN, Weber MB: What influences saturation? Estimating sample sizes in focus group research. Qual. Health Res. 2019; 29(10): 1483–1496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKannisto T: Trans*kinship, children’s rights, and the school. Int. J. Educ. Res. 2019; 94: 183–192. Publisher Full Text\n\nKar A, Mukherjee S, Ventriglio A, et al.: Attitude of Indian medical students towards homosexuality. East Asian Arch. Psychiatr. 2018; 28(2): 59–63. PubMed Abstract \| Publisher Full Text\n\nKaufman MR, Cornish F, Zimmerman RS, et al.: Health behavior change models for HIV prevention and AIDS care: Practical recommendations for a multi-level approach. J. Acquir. Immune Defic. Syndr. 2014; 66 Suppl 3(SUPPL.3): S250–S258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeuroghlian AS, Ard KL, Makadon HJ: Advancing health equity for lesbian, gay, bisexual and transgender (LGBT) people through sexual health education and LGBT-affirming health care environments. Sex. Health. 2017; 14(1): 119–122. PubMed Abstract \| Publisher Full Text\n\nKitzinger J: Qualitative research. Introducing focus groups.BMJ (Clinical Research Ed.).1995; 311(7000): 299–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKyngäs H, Mikkonen K, Kääriäinen M: The application of content analysis in nursing science research. Springer Nature; 2019.\n\nLaVaccare S, Diamant AL, Friedman J, et al.: Healthcare Experiences of Underrepresented Lesbian and Bisexual Women: A Focus Group Qualitative Study [published correction appears in Health Equity. 2018 Sep 20;2(1):233]. Health Equity. 2018; 2(1): 131–138. Published 2018 Jul 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLundin M: Building a framework to study the hetero norm in praxis. Int. J. Educ. Res. 2011; 50(5): 301–306. Publisher Full Text\n\nMacapagal K, Bhatia R, Greene GJ: Differences in Healthcare Access, Use, and Experiences Within a Community Sample of Racially Diverse Lesbian, Gay, Bisexual, Transgender, and Questioning Emerging Adults. LGBT health. 2016; 3(6): 434–442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacKenzie A, Suissa J, Robertson M: ‘From the margins’: Exploring the marginalisation, exclusion and oppression of overlooked groups in philosophy of education. Int. J. Educ. Res. 2020; 99: 101504. Publisher Full Text\n\nDr. Magnus CD , Dr. Lundin M : Challenging norms: University students’ views on heteronormativity as a matter of diversity and inclusion in initial teacher education. Int. J. Educ. Res. 2016; 79: 76–85. Publisher Full Text\n\nMcCann E, Brown M: The inclusion of LGBT+ health issues within undergraduate healthcare education and professional training programmes: A systematic review. Nurse Educ. Today. 2018; 64: 204–214. PubMed Abstract \| Publisher Full Text\n\nMosack KE, Brouwer AM, Petroll AE: Sexual Identity, Identity Disclosure, and Health Care Experiences: Is There Evidence for Differential Homophobia in Primary Care Practice?Women’s Health Issues.2013; 23(6): e341–e346. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker A, Bhugra D: Attitudes of British medical students towards male homosexuality. Sex. Relatsh. Ther. 2000; 15(2): 141–149. Publisher Full Text\n\nRenjith V, Yesodharan R, Noronha J, et al.: Qualitative methods in health care research. Int. J. Prev. Med. 2021; 12(1): IJPVM_321_19. Publisher Full Text\n\nSanchez NF, Rabatin J, Sanchez JP, et al.: Medical students’ ability to care for lesbian, gay, bisexual, and transgendered patients. Fam. Med. 2006; 38(1): 21–27. PubMed Abstract\n\nSequeira GM, Chakraborti C, Panunti BA: Integrating lesbian, gay, bisexual, and transgender (LGBT) content into undergraduate medical school curricula: A qualitative study. Ochsner J. 2012; 12(4): 379–382. PubMed Abstract\n\nVan Eeuwijk P, Angehrn Z: How to … Conduct a Focus Group Discussion (FGD). Methodological Manual. 2017.\n\nWahlen R, Bize R, Wang J, et al.: Medical students’ knowledge of and attitudes towards LGBT people and their health care needs: Impact of a lecture on LGBT health. PLoS One. 2020; 15(7 July): e0234743–e0234713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalker K, Arbour M, Waryold J: Educational Strategies to Help Students Provide Respectful Sexual and Reproductive Health Care for Lesbian, Gay, Bisexual, and Transgender Persons. J. Midwifery Womens Health. 2016; 61(6): 737–743. PubMed Abstract \| Publisher Full Text\n\nYang HC: Teaching lgbt+ health and gender education to future doctors: Implementation of case-based teaching. Int. J. Environ. Res. Public Health. 2021; 18(16). PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "221846", "date": "15 Dec 2023", "name": "Vimala Ramoo", "expertise": [ "Reviewer Expertise Nursing education", "Critical care nursing and Nursing Management and Leadership" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFeedback to authors\n\nDear authors, thank you for the opportunity to read and review this interesting research protocol. My suggestions/comments to further enhance the paper are:\n\nTitle: The title effectively conveys the primary focus and purpose of the study. It clearly indicates that the research aims to develop, implement, and evaluate a curriculum for health science professionals regarding LGBTQIA+ care. It's specific and informative, which is essential for attracting the right audience and communicating the study's objectives. Overall, the title is well-constructed and aligns with the research's objectives.\n\nScope of the Study: The scope of the study appears to be appropriately defined within the title and is further elaborated upon in the abstract and introduction sections of the research protocol. It includes three key phases: curriculum development, implementation, and testing, with the overarching goal of addressing healthcare disparities and enhancing LGBTQIA+ healthcare education for health science professionals.\nHowever, to further clarify the scope: In the introduction section, consider briefly mentioning the specific healthcare disciplines or professions within \"health science professionals\" that the curriculum is intended for (e.g., physicians, nurses, social workers) to provide a more precise context. I noted only the need for medical students and faculties is highlighted in the text. The importance for nurses and other healthcare professionals is not emphasized. Define who the healthcare providers are and use a consistent term either as healthcare providers or as healthcare professionals (healthcare science student mentioned in Figure 1). Avoid using them interchangeably, as this may confuse readers. Also, consider where students can be considered as providers or professionals.\nIt is noted also the authors had mentioned assessing knowledge of LGBTQIA+ health needs and sometimes knowledge of LGBTQIA+ care – please be consistent.\n\nResearch question\nThe research question requires minor refinement of the sentence structure, for example: How does an LGBTQIA+ curriculum impact healthcare providers' knowledge of LGBTQIA+ care?\nHowever, in the study the authors plan to assess the \"how\" using a quantitative approach, I would suggest rephrasing to: \"To what extent does an LGBTQIA+ curriculum quantitatively improve healthcare providers’ knowledge of LGBTQIA+ care?\"\nSuggestion for the secondary research question as this will be assessed using a pre-post approach: To assess the effectiveness of the curriculum in improving healthcare professionals’ knowledge of LGBTQIA+ care.\n\nPhase 1 – Development of curriculum\n\nThe curriculum development process should include a strong focus on cultural competence and sensitivity. This includes not only understanding healthcare needs but also addressing the unique cultural and ethical aspects of caring for LGBTQIA+ individuals.\nI would suggest that in addition to FGD and literature review, the authors should consider incorporating insights and expertise from healthcare professionals experienced in LGBTQIA+ healthcare. Experts can provide valuable guidance on best practices and ensure that the curriculum aligns with clinical reality.\nThe content of the expert validation of the curriculum requires further elaboration in terms of the panel's expertise, the number of experts involved and the specific analysis process to be carried out. Future details on these aspects would be valuable to ensure transparency and accuracy in the validation process. I recommend determining:\nThe composition of the expert panel, including the diversity of expertise among panel members (e.g., LGBTQIA+ healthcare, medical education, curriculum development). The total number of experts who will participate in the validation process. A clear description of the analysis process, indicating the criteria and guidelines against which the curriculum will be evaluated. How experts will provide feedback and recommendations and whether there will be a formalized assessment or evaluation system for assessing curriculum components.\n\nFGD participants: While the authors mention recruiting a representative number of self-identified LGBTQIA+ individuals, consider expanding efforts to ensure diversity within the sample. Mention whether they will actively seek participants from diverse backgrounds, age groups, and gender identities to capture a comprehensive range of perspectives and experiences.\nThe rationale for selecting a sample size of 10-15 participants is well-explained. The authors highlighted the importance of depth in the Focus Group Discussion (FGD) and subsequent analysis. It would be helpful to mention whether this sample size is consistent with previous research or guidelines in the field to further support the appropriateness of the sample size.\nExclusion criteria should not be the opposite of inclusion criteria, for example: “Any person who is not belonging to the LGBTQIA+ community” is not required.\n\nPhase II – Pilot study\n\nInsufficient information has been provided regarding the measurement instrument, and the questionnaire on knowledge. It is essential to include comprehensive details regarding the development or adaptation of the instrument, its content, the validation process, and reliability testing. The time interval between the pre-and post-test and its rationale.\n\nFigure 1 needs a more detailed description of the process. Figure 2 – not clear of the activity in Workshop I.\n\nDiscussions\nThe discussion touches on key points related to previous research and the potential impact of the curriculum. However, further elaboration, interpretation, and explicit connections between the current study and previous research would enhance the overall clarity and depth of the discussion. It would be advantageous to explicitly state how the present study seeks to build on or contribute to existing knowledge in this area. This will help readers understand the unique contributions of your research.\n\nAll the best\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11982", "date": "23 Jul 2024", "name": "RENJULAL YESODHARAN", "role": "Author Response", "response": "Thank you for reviewing the manuscript. We appreciate the suggestions given by you. As per your suggestions, the term ‘Medical students’ was changed to health care professionals. The research questions were modified based on the suggestions. Modifications in the figures were undertaken to accommodate these changes. We consistently used terminologies throughout the text, as per your suggestion. A few terminologies are replaced with more technical ones. Regarding the validation of the m content module, the authors have already included experienced healthcare professionals for their suggestions. The experts validate the content of the curriculum. We have also modified the discussions." } ] }, { "id": "221847", "date": "15 Dec 2023", "name": "Dr shubha Jayaram", "expertise": [ "Reviewer Expertise My areas of research are Medical Education", "Diabetes Mellitus", "Thanatochemistry", "Global Burden of 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\nThank you for your interest and intent in publishing this article titled, “Development, implementation, and testing of LGBTQIA+ care curriculum for health science professionals: Research protocol”. I appreciate the sincere efforts put together for the preparation of this manuscript .The topic is very relevant and the need of the hour. The following points should be addressed and the manuscript revised.\nTitle: Title should be suitably modified to replace the word “Curriculum “ , Curriculum is a broad terminology which is defined as follows :\n“Curriculum is what is taught in a given course or subject. Curriculum refers to an interactive system of instruction and learning with specific goals, contents, strategies, measurement, and resources”. The study is a research protocol for fulfilling the identified gaps in the health science curriculum to provide adequate care for LGBTQIA + subjects .Therefore I suggest that the word curriculum be modified as “ Development, implementation, and testing of a module for LGBTQIA+ care to be integrated in the curriculum for health science professionals: Research protocol\" .\n2.Abstract :\n\nA. “Additionally, healthcare professionals also find it difficult to care for LGBTQIA+ as very little is studied or heard about management.” This can be rephrased as “Additionally, healthcare professionals also find it difficult to care for LGBTQIA+ as they are not adequately trained regarding the management of these subjects”. B. Study outcome should be modified: to include\n\npolicy changing practices, not just improvement of knowledge. C. Keywords: Rationale for including SDG 3 and SDG -10? Sustainable Development Goal? As keywords as they nowhere appear in the manuscript and not discussed in the context, needs to be justified.\n3.Page 3: Background: “Minority stress”\n\nto be detailed in the context of health care education, the term QIA + in LGBTQIA+ is not explained. 4.Page 3: Background: April 2014: Reference to be provided. 5.Page 3: Background: “Although the Delhi High court……………….”, This sentence to be modified for clarity. 6.Page 3: Background: Delhi High Court 2009: –reference needed. 7.Page 4: para -1:- “Hetero sexist attitudes may result in Inaccurate Risk Assessment” This sentence to be justified and rewritten for clarity . 8.Page 4; para 2: Health institutions should make room for individuals –Needs to be rephrased. 9.Page 5 : Research Plan :- points b and c ,\nb. The present health science curriculum does not have a specific curriculum that deals with the management of LGBTQIA+ health problems. c. There is a lack of structured guidelines for managing the health conditions of LGBTQIA+ people.\n\nThese points should be highlighted and discussed adequately as the need for the study.\n10.Introduction to be rewritten, as it is not aligned with the objectives of the study. Research question is not aligned with the title of the topic and study objectives should be provided. What are ‘The gaps’ in the present curriculum / Gaps and lack of competency of medical professionals in communicating with and management of LGBTQIA+ subjects , whether it should be a part of the AETCOM modules of the present CBME (Competency Based Medical Education ) medical curriculum should be discussed in the background section. 11.Primary Research Question and Research Objectives are not clear. 12.In alignment with the research question, assessment of knowledge about LGBTQIA+ Care has to be included in the title. 13.Based on the previously published literature related to the needs of LGBTQIA+, it is assumed that……….:\n\nhas to be rephrased and modified. 14.A, B, C, points in the methodology have to be described and highlighted in the background section. 15.Phase -1 Flow chart diagram should include obtaining Institutional Ethics Committee clearance and taking consent from the subjects. 16.Methodology: Study design: Type of study design should be described; whether qualitative, quantitative, or Mixed methods study? 17.Page 6: Methodology: For Focussed Group Discussions (FGD) initials of principal investigator and moderator to be mentioned. 18.Page 6: Methodology: Inclusion criteria for FGD participants to be detailed, How were they approached? Sample size determination of participants? Criteria for inclusion?, Justification for age group of the LGBTQIA +subjects participating in FGD, Who are all the participants of FGD apart from LGBTQIA+ subjects to be clarified; whether a separate FGD would be held for faculty of health care professionals or whether they were also a part of the first FGD needs to be clarified and justified. 19.Page 6: Methodology: How the recording of the non-verbal communication was done during FGD to be described. 20.It is mentioned that the participants of the FGD will be compensated? Details required. 21.Box -1: FGD questions: Development of FGD lead questions, details required as to how the questions of FGD were validated? And whether any formal FGD interview guide was used needs to be described. 22.Page 4: Methodology: It is mentioned “To achieve this secondary research question, a structured knowledge-based questionnaire on LGBTQIA+ care will be administered to the health science students and faculty to assess their knowledge about LGBTQIA+ care.”\n\nThis structured questionnaire should be provided in the appendix/as annexure section. Whether Validity and Reliability of the questionnaire was tested, CVI and CVR were calculated;, whether I-CVI (Item level CVI )/ S-CVI (Scale level CVI ) were also calculated ? Validation process should be elaborated. 23.Page 7: Phase -11: Inclusion criteria for phase -2, age group of the health care students participating in the study to be given. 24.Page 8: How the Assessment of primary and secondary outcomes would be done; to be described.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11983", "date": "23 Jul 2024", "name": "RENJULAL YESODHARAN", "role": "Author Response", "response": "Thank you for reviewing the manuscript. Your expert suggestions are incorporated in version 2 of the manuscript. The title of the manuscript is changed, and the \"curriculum\" is changed to \"module\". The keywords of the study have been changed as per SDG3 and SDG10. As per your suggestion, 'minority stress is explained in the text. The details you asked about FGD will be published separately as an outcome paper; hence, it is not incorporated in this manuscript. The suggestion you mentioned will be considered while disseminating the findings of FGD. As per your suggestion, we have included more explanations for assessing the primary and secondary objectives." } ] } ]	1	https://f1000research.com/articles/12-1437
https://f1000research.com/articles/13-530/v1	24 May 24	{ "type": "Research Article", "title": "Association of maternal and cord vitamin B12 levels with anthropometry in term neonates born to malnourished mothers in coastal South India", "authors": [ "Sugapradha GR", "Ramesh Holla", "Poornima Manjrekar", "Suchetha Rao", "Sugapradha GR", "Ramesh Holla", "Poornima Manjrekar" ], "abstract": "Background Malnourished pregnant women are at increased risk of micronutrient deficiency. We assessed the vitamin B12 status in both malnourished and normally nourished pregnant women and their neonates. Additionally, we studied the association between maternal B12 levels, cord B12 levels and neonatal anthropometry.\n\nMethods This cross-sectional study enrolled 63 malnourished and 63 normally nourished mothers and neonates. Maternal and cord blood samples were collected at the time of delivery for estimation of vitamin B12 levels. Maternal and cord vitamin B12 levels were compared using the Mann–Whitney U test. Neonatal anthropometry was correlated with maternal and cord B12 levels using Spearman’s correlation. Data were analyzed using SPSS version 25.\n\nResults Mean maternal age was 26.58 yrs. The median cord B12 levels were lower than the maternal B12 levels. Maternal B12 levels showed a strong positive correlation with cord B12 levels (rho = 0.879; p < 0.001). Maternal (p < 0.001) and cord (p < 0.001) vitamin B12 levels were significantly lower in the malnourished group than in the normally nourished group. In malnourished group, 66.8% mothers and 95.2% neonates were Vitamin B12 deficient, whereas 1.5% mothers and 4.7% neonates were vitamin B12 deficient in normally nourished group. In the malnourished group, maternal B12 levels were positively correlated with birth weight (rho 0.363, p = 0.003) and length (rho 0.330, p =0.008), whereas cord B12 levels were positively correlated with birth weight in the normally nourished group. (rho 0.277 p= 0.028)\n\nConclusion High rates of vitamin B12 deficiency were observed in malnourished mothers and neonates. There was a positive correlation between birth weight, length, and maternal vitamin B12 levels in malnourished mothers. These findings emphasize the need to address maternal malnutrition and vitamin B12 deficiency to improve neonatal health.", "keywords": [ "Health", "low birth weight", "malnourished", "neonate", "pregnant women", "umbilical cord", "vitamin B12" ], "content": "Introduction\n\nVitamin B12, also known as cobalamin, is a micronutrient essential for cell growth and differentiation.1 This essential vitamin plays a crucial role in various biochemical processes, particularly in the synthesis of DNA, in conjunction with folic acid.2 Vitamin B12 is commonly found in animal-based food products such as dairy, fish, eggs, and poultry. Consuming these foods is an effective way to acquire vitamin B12 from the diet. While the vegetarian population is at a heightened risk of deficiency, it is also prevalent among non-vegetarians.3\n\nVitamin B12 deficiency is a major health concern worldwide. Pregnant women and young children are at an increased risk of facing this deficiency.4 Regional disparities in the occurrence of vitamin B12 deficiency throughout pregnancy have been observed, indicating varying levels of risks across different regions.5\n\nInadequacy of vitamin B12 in pregnancy has been linked to unfavourable pregnancy outcomes, including spontaneous abortions, stillbirth, neural tube defects, intrauterine growth restriction, low birth weight, and premature delivery.6–10 Deficiency of vitamin B12 is also implicated in increased risk of gestational diabetes mellitus.11 Adequate levels of vitamin B12 during early childhood promote growth and prevent cognitive impairment. A systematic review by Rogne et al. reported no clear linear relationship between birth weight and maternal vitamin B12 levels during pregnancy. However, deficiency was linked to a higher number of neonates born with low birth weight.12\n\nVitamin B12 deficient mothers may produce breast milk with insufficient levels of vitamin B12, and their exclusively breast-fed infants may develop symptoms in the postnatal period. These infants may have apathy, anorexia, irritability, failure to thrive, delayed or regression of milestones, pancytopenia, hypotonia, and seizures. Early symptoms may be nonspecific and pose challenges for identification. Vitamin B12 levels in expectant mothers are thought to influence the vitamin B12 status of the fetus and infant. The association between maternal, neonatal, and infant vitamin B12 levels has been heterogenous.13–17\n\nPregnant women experiencing malnutrition face a heightened risk of micronutrient deficiencies, pregnancy complications, and perinatal outcomes.18 There are limited data regarding the occurrence of vitamin B12 deficiency in malnourished expectant mothers and its impact on neonatal outcomes.\n\nWe aimed to assess the vitamin B12 status in malnourished and normally nourished pregnant women and to study the association between maternal B12 levels and cord B12 levels and neonatal anthropometry.\n\n\nMethods\n\nThis cross-sectional study was conducted in a community maternity facility affiliated with the Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India. The study was conducted between August 2020 and July 2021.\n\nThis cross-sectional study followed the standard Strengthening Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines.19 The reporting guidelines contain the completed STROBE checklist.20\n\nThe first group consisted of malnourished mothers and their neonates. The other group was comprised of normally nourished mothers and their neonates.\n\nAll term neonates born to normal and malnourished mothers under singleton pregnancies.\n\n\n\n(a) Pregnant women <18 years and > 35 years.\n\n(b) Pregnancy-induced hypertension/preeclampsia.\n\n(c) Maternal history of smoking and substance abuse.\n\n(d) Women with chorionic disease.\n\n(e) Women with diabetes mellitus.\n\n(f) Women with pre-existing cardiovascular hypertension or renal disorders.\n\n(g) Pregnant women receiving multivitamin supplements other than routine iron and folic acid.\n\n(h) Severe anemia.\n\n(i) Blood transfusions within 3 months.\n\n(j) Neonates with major congenital anomalies.\n\nThe sample size was determined based on a prior study by Finkelstein JL, et al., which reported the extent of vitamin B12 deficiency in South Indian pregnant women was 51%.21 With 95% confidence interval, 80% power, and a 5% level of significance. The sample size was calculated to be 63 for each group. A random sampling technique was used to reduce selection bias.\n\nMalnourished mother: A mother with a pre-pregnancy weight of less than 45 kg documented in antenatal records.22\n\nSmall for gestational age (SGA) is defined as birth weight < 10th percentile for gestation.23\n\nVitamin B12 level24\n\nNormal >300 pg/ml\n\nInsufficiency 200-300 pg/ml\n\nDeficiency <200 pg/ml\n\nOur research adhered to the ethical principles of world medical association Helsinki declaration of 1964 and later amendments. The study was carried out after obtaining permission from the Institutional Ethical Committee of KMC Mangalore, Manipal Academy of Higher Education (approval number IEC KMC MLR-08/2020/249 letter dated 20/08/2020), and necessary permissions were sought from hospital authorities. Based on the inclusion and exclusion criteria, suitable subjects were approached and provided with a participant information sheet. The purpose of the study was explained in their native language, and consent was obtained from those willing to participate. After obtaining consent, the participants were recruited for the study. Participation in the study was voluntary, and the complete anonymity of the research participants was maintained. We ensured confidentiality of the collected data.\n\nMaternal and neonatal details were obtained from antenatal and hospital records. Neonatal anthropometric measurements were performed by an investigator. Weight was measured to the nearest 0.01 kg using an electronic weighing machine, while the length of the newborn was estimated using an infantometer to the nearest to 0.1 cm, and the circumference of the head was calculated using a non-stretchable tape nearest to 0.1 cm. Based on birth weight and gestational age, the babies were categorized as Large, Small, and Appropriate for Gestational Age. Maternal and neonatal details were documented using a semi-structured pretested pro forma.\n\nAt the time of delivery, approximately 4 ml of maternal venous blood was collected in plain glass tubes, and 4 ml of blood from the umbilical cord was directly obtained via venipuncture of the umbilical vein. The specimens were then immediately placed on ice. Centrifugation was performed at a rate of 4000 revolutions per minute for 5 min. Subsequently, the plasma was separated and stored at -80°C pending analysis.\n\nSerum vitamin B12 levels were measured using a vitamin B12 ELISA kit (Epitope Diagnostics, San Diego, CA 92121, USA) at the Department of Biochemistry, KMC Centre for Basic Sciences, Bejai, Mangalore. In this assay, antibodies specific to vitamin B12 were bound to the surface of a microtiter plate. Standards or samples containing vitamin B12 and conjugated vitamin B12-peroxidase were added to the wells of a microtiter plate. Free vitamin B12 and enzyme labelled vitamin B12 compete for the binding sites on the antibodies. Following one hour of incubation at room temperature, the wells were washed with a diluted washing solution to eliminate any unbound materials. Subsequently, the substrate solution was added and incubated for 20 min, leading to the development of a blue color. This color development was inhibited by adding a stop solution, after which it turned yellow. Color intensity was measured photometrically at 450 nm. The concentration of vitamin B12 displayed an inverse relationship with the color intensity observed in the test sample.\n\nPercentage of maternal and cord B12 levels in normal, insufficient and deficient category.\n\nRelation between maternal and cord B12 level and neonatal anthropometry.\n\nThe collected data were entered into the Statistical Package for IBM (SPSS) Statistics for Windows version 25.0. Armonk, NY: IBM Corp. for analysis. Anthropometric and demographic data were expressed as means and standard deviations or proportions. Vitamin B12 levels are expressed as medians and interquartile ranges. Maternal and neonatal characteristics were compared in both groups using the independent sample t-test and chi-square test. Vitamin B12 levels were compared between groups using the Mann–Whitney U test. Correlations between maternal and cord B12 levels and neonatal anthropometries were assessed using the Spearman correlation test.\n\n\nResults\n\nOf the 126 pregnant women enrolled, 63 were malnourished and 63 were normal nourished. The mean age of the study subject was 26.58 (± 3.79) years. The mean pre-pregnancy weight was 48.68 (± 6.17) kg. Among these women, 75 (59.5%) were primigravidas. mixed diet was followed by 117 (92.9%) women, and all women consumed milk. Among the neonates, 64 (50.8%) were male and 62 (49.2) were female. The mean gestational age of the neonates was 38.39 (± 1.19) weeks. Mean birth weight was 2.77 (± 0.35) kg and 22 (17.5%) neonates belonged to small for gestation age (SGA) category. Maternal and neonatal characteristics are shown in Table 1.\n\n* Values expressed as %.\n\nBirth weight, length, and head circumference of neonates born to malnourished mothers were lower than those of neonates born to mothers with normal nourishment. Sixteen (25.4%) neonates of malnourished mother group were SGA. A comparison of the anthropometric parameters of neonates born to malnourished and normal-nourished mothers is shown in Table 2.\n\n* Values expressed as %.\n\nThe median cord B12 value was considerably lower than the maternal B12 value, as shown in Table 3. Both maternal and cord B12 levels were significantly lower in the malnourished group than in the normal-nourished group (Table 4). In malnourished group, 66.8% mothers and 95.2% neonates were vitamin B12 deficient. In normal nourished group, only 1.5% mothers and 4.7% neonates were deficient. The maternal and cord B12 levels in both groups are shown in Table 5. In the malnourished group, the maternal B12 level showed a positive correlation with birth weight (rho 0.363, p = 0.003) and length (rho 0.330, p = 0.008), whereas in the normally nourished group, the cord B12 level was positively correlated with birth weight (rho 0.277 p = 0.028) (Tables 6 and 7), respectively.\n\n\nDiscussion\n\nDuring gestation, the fetus depends entirely on maternal nutrition. Low levels of vitamin B12, primarily sourced from the mother at certain stages of gestation, can lead to adverse outcomes, such as low birth weight and early postnatal effects on cognitive development.25–27\n\nVitamin B12 deficiency is widespread globally and affects various populations. A study by Ramírez-Vélez et al in a sample representative of Columbian pregnant women, found that 18.6% of the population were deficient in Vitamin B12 and 41.3% exhibited insufficiency.28\n\nIn a nutritional intervention study that enrolled moderately malnourished Malawian mothers, 20.9% of the participants were deficient in Vitamin B12.29\n\nThere is a significant occurrence of Vitamin B12 deficiency in Indian women during pregnancy. Studies from Western and Northern parts of India have reported highest occurrence of Vitamin B12 deficiency during pregnancy, ranging from 70-74%.5\n\nA community based cross sectional study by Barney et al., enrolling pregnant women in rural South India, reported an extent of 55% Vitamin B12 deficiency. They found obesity and being in the first trimester of pregnancy to be independent risk factors.30\n\nA study conducted by Katre et al. in Pune, which recruited pregnant women during 17 weeks of gestation, indicated that approximately 80% of rural and 65% of urban women exhibited low Vitamin B12 status.31\n\nA prospective cohort study involving pregnant women in the age group of 18-45 years from urban hospitals of Bangalore reported low plasma vitamin B12 levels in 48.6% of women.32 In the present study, 66.8% women and 95.2% neonates of malnourished group exhibited vitamin B12 deficiency.\n\nA review by Sukumaran et al. suggested that even within non-vegetarian populations, vitamin B12 insufficiency during pregnancy is frequently observed.33 Similar results were observed in our study.\n\nConcentrations of vitamin B12 showed a declining trend from the first trimester to the third trimester, as reported by Sukumaran et al.33 Similar observations were reported by Sobowale et al. from Bangladesh.34 A cohort study involving healthy adolescent pregnant women from New York observed declining levels of maternal serum cobalamin levels from mid gestation to delivery. They found that maternal vitamin B12 concentrations at delivery were associated with infants’ vitamin B12 concentrations.35\n\nA study by Adaikalakoteswari et al. found that offspring of mothers with low B12 levels exhibited significantly lower levels of vitamin B12 compared to those born to mothers with normal levels.17\n\nA Study by Finkelstein e t al., which recruited pregnant women around 12 weeks gestation from South India, observed that vitamin B12 deficiency prevalent in around 63% women during early pregnancy.35 This deficiency served as predictor of neonatal vitamin B12 status. Specifically, lower maternal serum vitamin B12 levels are associated with a two-fold increased risk of neonatal vitamin B12 deficiency at birth.36\n\nYildizdas et al. conducted a study involving Turkish mothers and neonates, revealing alarmingly high rates of vitamin B12 deficiency, reaching 96.3%). They noted that micronutrient levels in the cord blood surpassed maternal levels. There was a negative correlation between birth weight, head circumference, and cord blood Cbl levels. Interestingly, maternal Cbl levels did not show any correlation with neonatal anthropometry.37 In the present study, cord B12 levels were lower than the maternal levels, and cord levels showed a positive correlation with maternal levels.\n\nIn a retrospective study by Yuan et al. in Eastern China involving 11,549 pregnant women, it was found that elevated maternal serum Vitamin B12 levels correlated with higher birth weight and an increased risk of newborn birth being large.7\n\nSukumar et al. concluded that there is no consistent relationship between vitamin B12 sufficiency and LBW.33 MAASTHI birth cohort study by Deepa et al. in South India found no association between vitamin B12 status and birth weight.32\n\nHay et al. reported a negative association between cord Cbl and birth weight. The heaviest babies had lower cord Cbl levels. A similar negative association was observed between birth length and head circumference also.38\n\nA secondary analysis of 709 Canadian mother newborn dyads by Tan et al. observed that maternal serum Vitamin B12 biomarkers did not show any association with birth weight or head circumference.39 In the present study, maternal B12 levels showed a positive correlation with birth weight (rho 0.363, p = 0.003) and length (rho 0.330, p = 0.008) but not with head circumference. In the normally nourished group, cord B12 levels showed a positive correlation with birth weight only (rho 0.277, p = 0.028).\n\nLimitations: This study was conducted in a tertiary care hospital, which may restrict the generalizability of its findings to a broader community. Maternal vitamin B12 levels measured only at the time of delivery may not capture fluctuations throughout pregnancy. A smaller sample size raises concerns regarding the applicability of the results. The presence of coexisting micronutrient deficiencies in malnourished women poses a risk of confounding bias in the interpretation of results.\n\nIn conclusion, the present study revealed high rates of vitamin B12 deficiency in malnourished mothers and their neonates. Additionally, There was a positive correlation between birth weight, length, and maternal vitamin B12 levels in malnourished mothers. These findings emphasize the need to address maternal malnutrition and vitamin B12 deficiency to improve neonatal health.\n\nInstitutional ethical approval was obtained from the ethics committee of Kasturba Medical College Mangalore (IEC KMC MLR-08/2020/249). Permission from the medical superintendent of the hospital was obtained prior to the study. A patient information sheet was provided and the purpose of the study was explained to the parents. Written informed consent was obtained from parents or legal guardians following the Helsinki Declaration. Participation in the study was voluntary, and the complete anonymity of the research participants was maintained. We ensured confidentiality of the collected data.", "appendix": "Data availability\n\nAssociation of maternal and cord vitamin B12 levels with anthropometry in term neonates born to malnourished mothers in Coastal South India. figshare. Dataset. https://doi.org/10.6084/m9.figshare.25622415. 20\n\nFigshare: STROBE checklist for ‘Association of maternal and cord vitamin B12 levels with anthropometry in term neonates born to malnourished mothers in Coastal South India.’ https://doi.org/10.6084/m9.figshare.25622415. 20\n\nThe data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank all neonates and their parents for participating in this study.\n\n\nReferences\n\nYajnik CS, Deshmukh US: Fetal programming: maternal nutrition and role of one-carbon metabolism. Rev. Endocr. Metab. Disord. 2012 Jun; 13(2): 121–127. PubMed Abstract \| Publisher Full Text\n\nSaravanan P, Sukumar N, Adaikalakoteswari A, et al.: Association of maternal vitamin B12 and folate levels in early pregnancy with gestational diabetes: a prospective UK cohort study (PRiDE study). Diabetologia. 2021 Oct; 64(10): 2170–2182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen LH, Miller JW, de Groot L , et al.: Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review. J. Nutr. 2018 Dec 1; 148(suppl_4): 1995S–2027S. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkelstein JL, Fothergill A, Venkatramanan S, et al.: Vitamin B12 supplementation during pregnancy for maternal and child health outcomes. Cochrane Database Syst. Rev. 2024 Jan 8; 2024(1): CD013823. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehere RV, Deshmukh AS, Otiv S, et al.: Maternal Vitamin B12 Status During Pregnancy and Its Association With Outcomes of Pregnancy and Health of the Offspring: A Systematic Review and Implications for Policy in India. Front. Endocrinol (Lausanne). 2021 Apr 12; 12: 619176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkelstein JL, Guillet R, Pressman EK, et al.: Vitamin B12 Status in Pregnant Adolescents and Their Infants. Nutrients. 2019 Feb 13; 11(2): 397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuan X, Han X, Zhou W, et al.: Association of folate and vitamin B12 imbalance with adverse pregnancy outcomes among 11,549 pregnant women: An observational cohort study. Front. Nutr. 2022; 9: 947118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzad C, Jat KR, Kaur J, et al.: Vitamin B12 status and neurodevelopmental delay in Indian infants: a hospital-based cross-sectional study. Paediatr. Int. Child Health. 2020 May; 40(2): 78–84. PubMed Abstract \| Publisher Full Text\n\nPepper MR, Black MM: B12 in fetal development. Semin. Cell Dev. Biol. 2011 Aug; 22(6): 619–623. PubMed Abstract \| Publisher Full Text\n\nGu Q, Li Y, Cui ZL, et al.: Homocysteine, folate, vitamin B12 and B6 in mothers of children with neural tube defects in Xinjiang, China. Acta Paediatr. 2012 Nov; 101(11): e486–e490. PubMed Abstract \| Publisher Full Text\n\nHe J, Jiang D, Cui X, et al.: Vitamin B12 status and folic acid/vitamin B12 related to the risk of gestational diabetes mellitus in pregnancy: a systematic review and meta-analysis of observational studies. BMC Pregnancy Childbirth. 2022 Jul 23; 22(1): 587. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRogne T, Tielemans MJ, Chong MF, et al.: Associations of Maternal Vitamin B12 Concentration in Pregnancy With the Risks of Preterm Birth and Low Birth Weight: A Systematic Review and Meta-Analysis of Individual Participant Data. Am. J. Epidemiol. 2017 Feb 1; 185(3): 212–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellows AL, Smith ER, Muhihi A, et al.: Micronutrient deficiencies among breastfeeding infants in Tanzania. Nutrients. 2017; 9: 1258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams AM, Stewart CP, Shahab-Ferdows S, et al.: Infant serum and maternal milk vitamin b-12 are positively correlated in Kenyan infant-mother dyads at 1–6 months postpartum, irrespective of infant feeding practice. J. Nutr. 2018; 148: 86–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoban S, Yilmaz Keskin E, Igde M: Association between maternal and infantile markers of cobalamin status during the first month post-delivery. Indian J. Pediatr. 2018; 85: 517–522. PubMed Abstract \| Publisher Full Text\n\nChebaya P, Karakochuk CD, March KM, et al.: Correlations between maternal, breast milk, and infant vitamin b12 concentrations among mother-infant dyads in Vancouver, Canada and Prey Veng, Cambodia: An exploratory analysis. Nutrients. 2017; 9: 270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdaikalakoteswari A, Vatish M, Lawson A, et al.: Low maternal vitamin B12 status is associated with lower cord blood HDL cholesterol in white Caucasians living in the UK. Nutrients. 2015; 7: 2401–2414. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGernand AD, Schulze KJ, Stewart CP, et al.: Micronutrient deficiencies in pregnancy worldwide: health effects and prevention. Nat. Rev. Endocrinol. 2016 May; 12(5): 274–289. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E , Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet. 2007 Oct 20; 370(9596): 1453–1457. PubMed Abstract \| Publisher Full Text\n\nRao Suchetha S, Sugapradha GR, Ramesh H, et al.: Association of maternal and cord vitamin B12 levels with anthropometry in term neonates born to malnourished mothers in Coastal South India. Dataset. figshare. 2024. Publisher Full Text\n\nFinkelstein JL, Kurpad AV, Thomas T, et al.: Vitamin B12 status in pregnant women and their infants in South India. Eur. J. Clin. Nutr. 2017 Sep; 71(9): 1046–1053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVervers MT, Antierens A, Sackl A, et al.: Which anthropometric indicators identify a pregnant woman as acutely malnourished and predict adverse birth outcomes in the humanitarian context? PLoS Curr. 2013 Jun 7; 5. Publisher Full Text\n\nWang KCW, James AL: Small for gestational age at term and adult lung function. Respirology. 2023 Feb; 28(2): 99–100. PubMed Abstract \| Publisher Full Text\n\nVargas-Uricoechea H, Nogueira JP, Pinzón-Fernández MV, et al.: Population Status of Vitamin B12 Values in the General Population and in Individuals with Type 2 Diabetes, in Southwestern Colombia. Nutrients. 2023 May 18; 15(10): 2357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMittal M, Bansal V, Jain R, et al.: Perturbing Status of Vitamin B12 in Indian Infants and Their Mothers. Food Nutr. Bull. 2017 Jun; 38(2): 209–215. PubMed Abstract \| Publisher Full Text\n\nHasbaoui BE, Mebrouk N, Saghir S, et al.: Vitamin B12 deficiency: Case report and review of literature. Pan Afr. Med. J. 2021; 38: 237.\n\nCruz-Rodríguez J, Díaz-López A, Canals-Sans J, et al.: Maternal Vitamin B12 Status during Pregnancy and Early Infant Neurodevelopment: The ECLIPSES Study. Nutrients. 2023 Mar 22; 15(6): 1529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamírez-Vélez R, Correa-Bautista JE, Martínez-Torres J, et al.: Vitamin B12 concentrations in pregnant Colombian women: analysis of nationwide data 2010. BMC Pregnancy Childbirth. 2016 Feb 1; 16: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlosz CM, Schaffner AA, Reaves SK, et al.: Effect of Nutritional Interventions on Micronutrient Status in Pregnant Malawian Women with Moderate Malnutrition: A Randomized, Controlled Trial. Nutrients. 2018; 10(7): 879. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarney AM, Abraham VJ, Danda S, et al.: Prevalence of Vitamin B12 Deficiency and Its Associated Risk Factors among Pregnant Women of Rural South India: A Community-based Cross-sectional Study. Indian J. Community Med. 2020 Oct-Dec; 45(4): 399–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatre P, Bhat D, Lubree H, et al.: Vitamin B12 and folic acid supplementation and plasma total homocysteine concentrations in pregnant Indian women with low B12 and high folate status. Asia Pac. J. Clin. Nutr. 2010; 19(3): 335–343. PubMed Abstract\n\nDeepa R, Mandal S, Van Schayck OCP, et al.: Vitamin B6 Levels and Impaired Folate Status but Not Vitamin B12 Associated with Low Birth Weight: Results from the MAASTHI Birth Cohort in South India. Nutrients. 2023 Apr 6; 15(7): 1793. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSukumar N, Rafnsson SB, Kandala NB, et al.: Prevalence of vitamin B-12 insufficiency during pregnancy and its effect on offspring birth weight: a systematic review and meta-analysis. Am. J. Clin. Nutr. 2016 May; 103(5): 1232–1251. PubMed Abstract \| Publisher Full Text\n\nSobowale OI, Khan MR, Roy AK, et al.: Prevalence and Risk Factors of Vitamin B12 Deficiency among Pregnant Women in Rural Bangladesh. Nutrients. 2022 May 10; 14(10): 1993. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkelstein JL, Guillet R, Pressman EK, et al.: Vitamin B12 Status in Pregnant Adolescents and Their Infants. Nutrients. 2019 Feb 13; 11(2): 397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkelstein JL, Fothergill A, Krisher JT, et al.: Maternal vitamin B12 deficiency and perinatal outcomes in southern India. PLoS One. 2021 Apr 6; 16(4): e0248145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYapicioglu Yildizdas H, Erdogan BG, Tepe T, et al.: Birth weight, head circumference, and length of newborns are unaffected by maternal levels of vitamin B12 and folate. Nutr. Res. 2022 Aug; 104: 101–107. PubMed Abstract \| Publisher Full Text\n\nHay G, Clausen T, Whitelaw A, et al.: Maternal Folate and Cobalamin Status Predicts Vitamin Status in Newborns and 6-Month-Old Infants. J. Nutr. 2010; 140: 557–564. PubMed Abstract \| Publisher Full Text\n\nTan A, Sinclair G, Mattman A, et al.: Maternal vitamin B12 status in early pregnancy and its association with birth outcomes in Canadian mother-newborn Dyads. Br. J. Nutr. 2021 Dec 28; 126(12): 1823–1831. PubMed Abstract \| Publisher Full Text" }	[ { "id": "289097", "date": "22 Jun 2024", "name": "Rathika Damodara Shenoy", "expertise": [ "Reviewer Expertise Perinatology", "Metabolic disorders", "Dysmorphology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe cross-sectional study compares the maternal and cord blood B12 levels in normally nourished and malnourished mothers at term. The authors report lower maternal and cord blood B12 values in the malnourished group and a strong positive correlation between the measures. The correlations between B12 values and newborn anthropometry are weak to none. The results reveal that a significant proportion, about 53%, of women in the study population have insufficient or deficient B12 status despite a mixed diet. It may be worthwhile to compare the maternal demography of the two groups (like Table 2 on newborn data). The authors can investigate if maternal BMI and B12 correlation will provide additional information. The limitations are listed by the authors. The main takeaway from the study is the high prevalence of maternal B12 deficiency in a coastal fish-eating community. Food taboos targeting pregnant and postpartum women are widely prevalent in India and South Asian countries.1,2 The authors should investigate qualitative and quantitative diet information, the coexistence of iron deficiency, and the clinical relevance in future studies with a larger sample size.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "12028", "date": "23 Jul 2024", "name": "SUCHETHA S RAO", "role": "Author Response", "response": "Review comment : The cross-sectional study compares the maternal and cord blood B12 levels in normally nourished and malnourished mothers at term. The authors report lower maternal and cord blood B12 values in the malnourished group and a strong positive correlation between the measures. The correlations between B12 values and newborn anthropometry are weak to none. The results reveal that a significant proportion, about 53%, of women in the study population have insufficient or deficient B12 status despite a mixed diet. It may be worthwhile to compare the maternal demography of the two groups (like Table 2 on newborn data). Author reply : Thank you for reviewing our article and giving valuable suggestions. We have modified the article as per your suggestions. We have compared the maternal demography and details are added to table 2 along with newborn data Review comment : The authors can investigate if maternal BMI and B12 correlation will provide additional information. Author reply : Spearman correlation test for BMI and Vitamin B12 level was done. There was a positive correlation between the two measures. ( rho 0.590 , p <0.001) This observation has been added to result. Review comment :The limitations are listed by the authors. The main takeaway from the study is the high prevalence of maternal B12 deficiency in a coastal fish-eating community. Food taboos targeting pregnant and postpartum women are widely prevalent in India and South Asian countries.1,2 Author reply : Thank you for highlighting the significance of food taboos for possible contribution towards maternal Vitamin B12 deficiency along with references. We have added this to discussion. Review comment The authors should investigate qualitative and quantitative diet information, the coexistence of iron deficiency, and the clinical relevance in future studies with a larger sample size. Author reply We will plan for future studies" } ] }, { "id": "289090", "date": "29 Jun 2024", "name": "Noor Rohmah Mayasari", "expertise": [ "Reviewer Expertise Nutritional epidemiology", "anemia", "food security" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: The study aimed to assess vitamin B12 status in malnourished and normally nourished pregnant women and study the association between maternal B12 level and cord B12 labels and neonatal anthropometry. However, the paper hasn't discussed the findings related to the aim.\nIntroduction:\nPlease modify the introduction structure (the paragraph doesn't have the main sentence of each). Some paragraphs are too short (less than three sentences)\nMethods:\nPlease clarify the study design, is there a case-control or cross-sectional study? Why exclude severe anaemia, is there any specific reason? Malnourished is define as a pre-pregnancy body weight less than 45kg. Why use body weight rather than pre-pregnancy body mass index?\nResults:\nExplain the reason why in the table presentation authors use different display outcomes (table 2 (mean and SD), table 3 (median and interquartile range). Table 2 expresses as (%) or (mean and SD)? Tables 2-5can be merged as 1 table and Tables 6-7 can be merged as 1 table. From the study authors can analyze RR or OR\nDiscussion:\nWhat Is the key finding of this study? Please modify the discussion structure (the paragraph doesn't have the main sentence of each). Some paragraphs are too short (less than three sentences). The data shows that maternal vitamin B12 positively correlated with Birth weight and length among malnourished women. However, this correlation is not significant with cord vitamin B12, what is that mean and implication? Are there any maternal characteristics that effect the main outcome as confounding factors?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "12029", "date": "23 Jul 2024", "name": "SUCHETHA S RAO", "role": "Author Response", "response": "Reviewer Comment: General comments: The study aimed to assess vitamin B12 status in malnourished and normally nourished pregnant women and study the association between maternal B12 level and cord B12 labels and neonatal anthropometry. However, the paper hasn't discussed the findings related to the aim. ------------------ Author reply : Thank you for reviewing our article and giving valuable suggestions. We have modified the article as per your suggestions. We have added additional data related to aim. ------------------ Reviewer Comment: Introduction: 1.Please modify the introduction structure (the paragraph doesn't have the main sentence of each). Some paragraphs are too short (less than three sentences) ------------------ Author reply : Introduction has been modified as per the suggestion First paragraph- Role of Vitamin B12 in health , sources Second paragraph- Deficiency and health burden , prevalence and regional disparities of deficiency Third paragraph -Vitamin B12 deficiency adverse outcomes in pregnancy , and effect of maternal deficiency on child health Fourth paragraph - Need for the present study and aims of the study ------------------ Reviewer Comment: Methods 1. Please clarify the study design, is there a case-control or cross-sectional study? Author reply Cross sectional study 2. Why exclude severe anaemia, is there any specific reason? Author reply Severe anemia itself can lead to low birth weight babies. 3 .Malnourished is define as a pre-pregnancy body weight less than 45kg. Why use body weight rather than pre-pregnancy body mass index? ------------------ Author reply : Antenatal records had pre pregnancy weight documented not BMI ,hence we chose weight criteria for defining malnourished mother ------------------ Reviewer Comment: Results: 1.Explain the reason why in the table presentation authors use different display outcomes (table 2 (mean and SD), table 3 (median and interquartile range). ------------------ Author reply : Table 2 mentions about anthropometric measures which followed normal distribution , hence we used mean and SD Table 3 mentions about vitamin B12 levels which did not follow normal distribution , hence used Median and Interquartile range ------------------ Reviewer Comment: 2.Table 2 expresses as (%) or (mean and SD)? ------------------ Author reply : Expressed as Mean and SD or as (%). values marked * are expressed as (%) ------------------ Reviewer Comment: 3.Tables 2-5 can be merged as 1 table and Tables 6-7 can be merged as 1 table. ------------------ Author reply : We have merged table 4 and 5 as one table ( new table no 4 ) We have merged table 6 and 7 as one table ( new table no 5 ) ------------------ Reviewer Comment: 4.From the study authors can analyze RR or OR ------------------ Author reply : We have calculated OR. OR for malnourished mothers to have vitamin B12 deficiency is 160 ( 95% CI 39.43, 649.27) ------------------ Reviewer Comment: Discussion: 1.What Is the key finding of this study? ------------------ Author reply : A significant proportion, about 53%, of women in the study population have insufficient or deficient B12 status despite a mixed diet The median cord B12 value was considerably lower than the maternal B12 value and there was a strong positive correlation between maternal and cord B12 values Both maternal and cord B12 levels were significantly lower in the malnourished group than in the normal-nourished group In malnourished group , maternal vitamin B12 positively correlated with Birth weight and length. Cord B12 showed no correlation with neonatal anthropometry ------------------ Reviewer Comment: 2. Please modify the discussion structure (the paragraph doesn't have the main sentence of each). Some paragraphs are too short (less than three sentences). ------------------ Author reply : We have modified the discussion as per the suggestion ------------------ Reviewer Comment: 3. The data shows that maternal vitamin B12 positively correlated with Birth weight and length among malnourished women. However, this correlation is not significant with cord vitamin B12, what is that mean and implication? ------------------ Author reply : Maternal Vitamin B12 levels during pregnancy may directly influence growth of fetus , leading to higher birth weight and increased length. This implies that adequate maternal Vitamin B12 level is crucial for fetal growth. Low levels of maternal vitamin B12, at certain stages of gestation, can lead to adverse outcomes, such as low birth weight and shorter neonates. Previous studies have reported declining concentrations of maternal vitamin B12 from first trimester to the third trimester. Cord blood Vitamin B12 levels reflect status of Vitamin B12 of neonate at birth. Absence of correlation with cord Vitamin B12 means that Cord Vitamin B12 may not be a direct measure of ongoing maternal B12 influence during pregnancy. Critical periods of Vitamin B12 influence may occur at certain stages of gestation which may not be captured by Cord blood B12 value. This implies the need to maintain adequate maternal Vitamin B12 levels throughout pregnancy for better neonatal outcome. ------------------ Reviewer Comment: 4. Are there any maternal characteristics that effect the main outcome as confounding factors? ------------------ Author reply : Co existing micronutrient deficiencies in malnourished mothers may effect the main outcome as confounding factors. This has been mentioned in limitations. ------------------" } ] } ]	1	https://f1000research.com/articles/13-530
https://f1000research.com/articles/13-823/v1	23 Jul 24	{ "type": "Case Report", "title": "Case Report: Gallstone ileus management - Case report and review of the literature", "authors": [ "Rania Dallagi", "Wael Ferjaoui", "Ahmed Omry", "Hager Behi", "Med Bachir Khalifa", "Rania Dallagi", "Wael Ferjaoui", "Hager Behi", "Med Bachir Khalifa" ], "abstract": "Gallstone ileus, a rare complication of cholelithiasis, presents significant morbidity and mortality challenges, with no established consensus on optimal management. This study aimed to highlight the complexities surrounding its occurrence and emphasize the need for tailored therapeutic strategies. An 88-year-old female, with a history of type 2 diabetes mellitus presented with diffuse abdominal pain and vomiting. Clinical evaluation revealed signs of small-bowel obstruction. Radiological assessments, including Computed Tomography CT) scans, confirmed biliary ileus, showcasing a sizable gallstone causing subacute obstruction. Emergency surgery involving enterolithotomy was performed, successfully addressing the immediate concerns. Postoperative follow-up demonstrated a one-year asymptomatic period, emphasizing the effectiveness of the chosen intervention. Gallstone ileus typically follows acute cholecystitis, leading to gallstone erosion and fistula formation commonly in the duodenum. Diagnosis is challenging because of nonspecific symptoms, necessitating a high index of suspicion. Computed tomography (CT) plays a pivotal role in accurate and rapid diagnoses. This study delves into the intricate details of gallstone ileus presentation, complications, and the debate surrounding optimal surgical management, acknowledging the effectiveness of the two-stage procedure and emerging laparoscopic approaches. This case provides valuable insights into the intricate facets of gallstone ileus and emphasizes the need for individualized treatment strategies. Successful management, as demonstrated in our case, underscores the importance of considering patient-specific factors when choosing between the surgical approaches. This study supports recent reports advocating for laparoscopic interventions, encouraging further exploration of evolving therapeutic modalities for gallstone ileus.", "keywords": [ "Gallstone ileus", "Cholecysto-duodenal fistula", "Small bowel obstruction", "Case report" ], "content": "Introduction\n\nSmall bowel obstruction triggered by gallstones is a rare complication of cholelithiasis.1,2 It is defined as a mechanical obstruction of the intestine caused by the entrapment of a sizable gallstone that perforates the gallbladder wall, leading to the formation of a biliary digestive fistula.1 This condition arises in approximately 0.3 to 0.5% of individuals with cholelithiasis.3,4 It is associated with comparatively elevated rates of morbidity and mortality.1,2 There is no consensus regarding the optimal management of gallstone ileus.5,6 The present case involved a subacute small bowel obstruction caused by a gallstone, which was addressed through open enterolithotomy. Through this case report and literature review, our aim was to underscore the conditions leading to the development of this pathology, as well as the challenges associated with clinical and paraclinical diagnosis and therapy.\n\n\nCase presentation\n\nWe report the case of an 88-year-old female patient with a history of type 2 diabetes on oral antidiabetic medication and no surgical history. The patient presented to the emergency department with diffuse abdominal pain accompanied by early postprandial vomiting and cessation of bowel movements and gas, which persisted for the last two days. She denied having fever, jaundice, melena, discolored urine, or abdominal trauma preceding the onset of symptoms.\n\nPhysical examination revealed that the patient was hemodynamically stable without jaundice or fever. Abdominal examination revealed a distended tympanic abdomen with tenderness on palpation in the suprapubic and periumbilical regions. The hernial orifices were free, and rectal examination indicated normal-colored stools.\n\nLaboratory results indicated a biological inflammatory syndrome with a white blood cell count of 15,000 cells/mm3 and C-reactive protein (CRP) level of 100 mg/L. The remaining laboratory findings, including liver function test results, were within the normal range.\n\nAn unprepared abdomen showed small bowel air-fluid levels with an approximately 4 cm round pelvic opacity (Figure 1). Further exploration using an abdominal CT scan revealed a dilated proximal small bowel with a maximum measured diameter of 40 mm upstream of a transitional level consisting of a distended loop with a flattened appearance. At this level, an intraluminal stone measuring 3 cm in diameter was observed. In addition, there were no radiological signs of intestinal distress. The scan also indicated a multilithiatic gallbladder with a cholecystoduodenal fistula confirmed by an intra-vesicular air bubble (Figure 2). Furthermore, no intra-abdominal fluid was found. A diagnosis of biliary ileus was made.\n\nAfter admission, the patient underwent a hydration and analgesia protocol. A urinary catheter and nasogastric tube for gentle aspiration were inserted, yielding a fecaloid-looking fluid. Subsequently, the patient underwent emergency surgery via a midline approach.\n\nIntraoperative exploration revealed a dilated and viable proximal small bowel upstream of an intraluminal stone located 1 m 30 cm from the ligament of Treitz and 1 m 50 cm from the ileocecal valve (Figure 3). The distal small bowel and colon were flat and normal, respectively. An inflammatory mass involving the gallbladder, duodenum, right colic angle, and omentum was identified. An enterotomy was performed in a non-inflammatory healthy area, through which the stone was extracted (Figure 4). The opening was closed in two layers using Vicryl 3/0. We decided to postpone any additional procedures, such as cholecystectomy or closure of the cholecystoduodenal fistula, given the patient’s age and intraoperative findings. The intervention included peritoneal lavage using warm saline and drainage of the Douglas pouch. Postoperative recovery was uneventful, with the patient being allowed a regular diet from postoperative day 1 and discharged on postoperative day 4. The patient remained asymptomatic during one year of follow-up at the outpatient clinic, and radiological control showed spontaneous closure of the biliary digestive fistula.\n\n\nDiscussion\n\nGallstone ileus is a rare complication of cholelithiasis, occurring in less than 0.5% of patients who present with mechanical small-bowel obstruction.2,3,7 It has a higher incidence in females and older individuals, as in our case.1,8\n\nGallstone ileus (GI) is typically preceded by an initial acute cholecystitis.1,2,9 Inflammation affecting the gallbladder and its adjacent structures, compounded by the pressure exerted by the gallstone, can result in erosion of the gallbladder wall, ultimately leading to fistula formation.1,10 Fistulas connecting the gallbladder and gastrointestinal tract commonly manifest in the duodenum owing to their proximity.4 Other regions of the gastrointestinal tract may also be implicated, including the stomach, small intestine, and transverse colon.1,11 Because of their narrow lumens, the terminal ileum and ileocecal valve are frequently the primary locations of gallstone ileus.1 Gallstones causing small bowel obstruction (SBO) typically exceeding 2.5 cm.1,4,6 In our case, gallstone ileus manifested in the jejunum due to the considerable size of the gallstone (4 cm).\n\nDiagnosis is often challenging because of nonspecific or incomplete symptoms, such as intense abdominal pain, nausea, vomiting, inability to pass gas or stool, and hematochezia.6 Physical examinations may reveal abdominal distension and tenderness, decreased or absent bowel sounds, jaundice, and signs of dehydration.6\n\nBiochemical markers may appear normal or lack specificity, potentially featuring findings such as leukocytosis and abnormalities in electrolyte levels, as observed in our patient.9\n\nCT scans exhibit high sensitivity and specificity in diagnosing gallstone ileus, with rates of 93% and 100%, respectively.1,10 Therefore, a heightened level of suspicion is essential in individuals with a history of gallstones who present with bowel obstruction.9 Rigler’s triad delineates classical imaging features indicative of gallstone ileus: (1) intestinal obstruction, (2) pneumobilia, (3) gallstone within the intestinal lumen, and more recently, a change in the position of the gallstone observed in serial imaging.3,9\n\nThere is no consensus regarding the surgical management of gallstone ileus.1,5,12 Laparotomy is commonly considered to be the preferred method.1 The treatment objective is to alleviate obstruction by focusing on gallstone extraction.9 Surgical options encompass a one-stage approach involving enterolithotomy, cholecystectomy, and fistula closure or a two-stage procedure with enterolithotomy, cholecystectomy, and subsequent fistula closure.1 In our case, we performed only an enterotomy with stone extraction, considering the patient’s condition and the highly inflammatory mass formed by the gallbladder, thereby reducing postoperative morbidity. The one-stage procedure helps prevent the recurrence of gallstone ileus, with rates ranging from 8 to 33%.1 Nevertheless, this approach is technically challenging and associated with increased morbidity and mortality, particularly in older patients with comorbidities.1,6,8 In contrast, the two-stage procedure is more straightforward and requires a shorter operative time.1 As a result, it has emerged as a safer option for patients with compromised general conditions, dehydration, sepsis, and shock.1 Recently, laparoscopic management has been reported cases of laparoscopic management.1 This approach has proven to be both effective and safe, particularly in the context of a two-stage procedure.\n\nIn conclusion, we present a novel case of gallstone ileus that was successfully treated using a two-stage procedure. The choice of surgical approach should be tailored to factors such as the patient’s overall health, hemodynamic status, local conditions, and surgeon’s expertise.1\n\n\nConclusion\n\nGallstone ileus (GI) is a rare complication of cholelithiasis, particularly in females and older individuals.5,9 Our case, successfully treated with an enterotomy, highlights the challenges in diagnosis and emphasizes the importance of individualized surgical approaches based on patient characteristics. The choice between one- and two-stage procedures depends on the overall health of the patient.1 Recent reports have suggested that laparoscopic management is a safe and effective option. This case contributes to our understanding of gallstone ileus and underscores the need for tailored treatment.\n\nEthical approval were not required. Written informed consent for the publication of this case and the associated images was obtained from the patient.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nBeji H, Chtourou MF, Zribi S, et al.: Gallstone ileus: A case report and review of the literature. Int. J. Surg. Case Rep. 2023 May; 106: 108221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShrestha N, Mishra A, Ghimire R: An unusual case of subacute small bowel obstruction - Gallstone ileus. Int. J. Surg. Case Rep. 2022 Mar; 92: 106820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalgowski T, Pawlus J, Malanowska E, et al.: Gallstone ileus – case description and literature review.\n\nSuro-Santos Y, Serrato-Ruíz JA, Fuentes-Hernández JE, et al.: Biliary ileus an uncommon cause of intestinal occlusion: case report. HGMX. 2023 Feb 8; 86(1): 9676. Publisher Full Text\n\nVera-Mansilla C, Sanchez-Gollarte A, Matias B, et al.: Surgical Treatment of Gallstone Ileus: Less Is More. Visc. Med. 2022; 38(1): 72–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInukai K: Gallstone ileus: a review. BMJ Open Gastroenterol. 2019 Nov; 6(1): e000344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuñoz-Leija MA, Alemán-Jiménez MC, Quiroga-Garza A, et al.: Gallstone Ileus in a Young Patient: A Clinical Case Report and Literature Review. Cureus. 2023 Jan 3 [cited 2024 Jan 1]; 15: e33291. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nMorosin T, De Robles MSB, Putnis S: Gallstone Ileus: An Unusual Cause of Intestinal Obstruction. Cureus. 2020 Mar 15 [cited 2024 Jan 1]; 12: e7284. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nBoudou M, Jabi R, Maamar K, et al.: A febrile occlusion revealing a biliary ileus. Int. J. Surg. Case Rep. 2022 May; 94: 107113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPichardo J, Zapata J, Echavarría R, et al.: Gallstone Ileus With Cholecystoenteric Fistula in an Elderly Female: A Case Report. Cureus. 2023 Apr 3 [cited 2024 Jan 1]; 15: e37077. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nKirchmayr W, Mühlmann G, Zitt M, et al.: GALLSTONE ILEUS: RARE AND STILL CONTROVERSIAL. ANZ J. Surg. 2005 Apr; 75(4): 234–238. PubMed Abstract \| Publisher Full Text\n\nDreifuss NH, Schlottmann F, Cubisino A, et al.: Totally laparoscopic resolution of gallstone ileus: A case report. Int. J. Surg. Case Rep. 2022 Jan; 90: 106682. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "311699", "date": "21 Aug 2024", "name": "Federico Seifarth", "expertise": [ "Reviewer Expertise Minimally invasive surgery", "pediatric surgery." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report describes an elderly female patient suffering from gallstone ileus, a rare complication of cholelithiasis. The authors describe the history, clinical presentation, diagnostics and therapeutic considerations of treatment and the surgical procedure. In general, surgery is the accepted treatment of choice for such cases. I would therefore not postulate a \"lack of consensus of management\" but rather mention the challenges in diagnosis and discuss the different surgical technical options. The images are appropriate. Figure 2 shows pneumobilia which is present in 30-60% of all patients and should also be called as such. The authors should also discuss the diagnostic value of ultrasound and HIDA scans in the discussion and mention the rare event of an impaction of a large gallstone within the pyloric channel (Bouveret syndrome). This case does not represent a novel case of gallstone ileus (last paragraph) but discusses presentation, workup and therapy of this rare but well described pathology in elderly women.\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": "319563", "date": "26 Sep 2024", "name": "Sarthak Uttam", "expertise": [ "Reviewer Expertise general surgery", "GI 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\nHello dear researcher, This case report is well written and structured but i have a few queries which i would love to be addressed. 1. usually patients with gall stone ileus present with past history of multiple episodes of recurring cholecystitic pain and discomfort. Was it the case here ? 2. the ct image of pnemobilia appears a little dubious to me, can you provide a cross sectional axial image to delineate the air in gall bladder and the enteral stone ? and is there a cholecystoduodenal fistule that can be spotted in ct scan ? 3. was an ultrsonography of gall bladder soughted before surgery ? usg has better sensitivity for gall stones that ct scan. 4. was the enterotomy for stone extraction done proximal or distal to the site of stone impaction ? and was the remaining bowel thoroughly inspected for other stones that might have been missed in imaging because you mentioned GB was 'multilithiatic'. 5. it there were stones in gall bladder there is apparent risk for recurrences. what was the plan for management of remnant stones ? how was the patient followed ? and was there a scope of getting endoscopy done during the follow up to look for fistula healing ? 6. you mentioned in the management that patient recovered with spontaneous closure of the fistula, but in discussion you mention that a two staged procedure was followed. this is highly contradicting and please mention the details of second procedure done. These questions i feel are important in terms of patient follow up and in preventing recurrences in this old age lady who is a known diabetic. Mention of these details would make your report more complete and inclusive. kindly refer to the citations attached. thankyou.\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? 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/13-823
https://f1000research.com/articles/13-820/v1	22 Jul 24	{ "type": "Research Article", "title": "Impact of Inaccurate Clinical Coding on Financial Outcome: A Study in a local hospital in Najran, Saudi Arabia.", "authors": [ "Salem Albagmi" ], "abstract": "Background Coding in medical procedures is crucial for patients, and errors made by hospital administration during the coding process can have an impact on both the financial results and the course of therapy. The present study aims to assess the accuracy of diagnostic and procedural codes as recorded by the hospital’s coders and to also evaluate their impact on the hospital’s revenue.\n\nMethods In a local hospital in Najran, Saudi Arabia, a cross-sectional observational analysis was conducted on patients with a clinical coder. The percentage of precision and error following the re-coding of cases was calculated using a statistical analysis.\n\nResults Primary diagnosis was incorrectly coded in 57 (26 per cent) records, and secondary diagnosis was incorrectly coded in 21 (9.9 per cent) records. Inaccurate medical labelling has been seen in emergency rooms, operating rooms, and gynaecology facilities.\n\nDiscussion The percentage of records with the most incorrect coding was found to be 16 (7.5 per cent) in the emergency room, 10 (4.7 per cent) in the surgical clinic, and 5 (2.3 per cent) in the gynaecology/OBS clinic. Six (2.8 per cent) records in the private clinic had inaccurate secondary diagnoses, followed by four (1.9 per cent) and two (1 per cent) records in nephrology.\n\nConclusion The percentage of inaccurate clinical codes in primary diagnoses reached (26.8 per cent) and the percentage of incorrect clinical codes in secondary diagnoses reached (9.9 per cent).", "keywords": [ "Clinical Coding", "Diagnosis", "Hospitals" ], "content": "Introduction\n\nClinical coders transform clinical terminologies into coded language, which represents a system of figures and letters. Diagnostic and procedural codes are major components of the coding process. In Saudi Arabia, clinical coders use the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Australian Modification (ICD-10-AM) for illnesses and health issues, and the Australian Classification of Health Interventions (ACHI) for procedures.1 These two systems aid medical professionals in the creation of a standardized coding for illnesses, procedures, medical consultations, and causative factors for accidents. Moreover, hospital reimbursement, audits, studies, planning, and administration also rely on coded data.2\n\nThe cost of care and income are calculated using two different financial systems i.e. Diagnostic Related Group (DRG) and Case Mix Index (CMI). DRG, developed in the United States, was then adopted by other nations. Its main purpose is to collect data on the patients treated, and the amount earned by the hospital. With the assignment of coding as per the International Classification of Diseases, Ninth Revision, and Clinical Modification (ICD-9-CM) to the illness and procedures, DRG categorizes the patients based on their diagnosis and thus monitors the fact that all the patients are charged the same amount for a particular procedure. The Centers for Medicare and Medicaid Services (CMS) implemented the Inpatient Prospective Payment System, based on DRG.1,2\n\nCMI classifies patients according to their clinical characteristics and acts as a provider payment tool that helps monitor hospital resources and income. Despite the original principle for its development was the calculation of hospital payments in terms of patient care, it has recently gained momentum as a platform for public reporting of quality indicators and costs for disease severity (Indicator or costs ÷ CMI of the medical centre). This helps in making comparisons among various medical centres. However, the authenticity of CMI may be affected by the accuracy of the documentation skills of the physician and the coding expertise of the coder using ICD-9-CM codes.3\n\nAccuracy in the coding system is very crucial as the entry of a single wrong code can result in a domino effect which might end in affecting the hospital’s income. Thus, the accuracy of clinical codes which are eventually used for payment, health statistics, research, and planning purposes, requires thorough verification.4,5 The present study aimed to assess the accuracy of diagnostic and procedural codes as recorded by the hospital’s coders and to also evaluate their impact on the hospital’s revenue.\n\n\nMethods\n\nThe study was reviewed by the institutional review board which provided an exemption due to the retrospective nature of the study. The coded cases, from a local tertiary care hospital in Najran, Saudi Arabia, with complete patient medical records consisting of diagnosis, procedures done, and the bills paid (either by the patient or the insurance company) were included in the study on a non-probability consecutive basis. The coding was done using ICD-10-AM, and ACHI.\n\nThe reviewing process, done by the HIM department staff, involved all the health records as well as the inpatient fact sheet (part of the health record) that is used by clinical coders to write diagnoses and procedural codes. The recoding was done using the ICD-10-AM, and ACHI which was the same as used by the hospital’s coders. Following the reviewing and recoding processes, the differences were documented in a special form and then the codes were compared to evaluate the accuracy of the initial codes. The comparison also comprised an assessment of the change in the total charges incurred based on the change in the code (if any).\n\nThe approval was obtained from the Institutional Review Board of Prince Sultan Military College of Health Sciences (IRB-2021-HIM-023) on June 6, 2023. The study was conducted in accordance with the declaration of Helsinki.\n\nThe inaccuracy observed during the reviewing and recoding process was determined using the following formula:\n\nFurthermore, the total financial impact that might have been incurred due to inaccurate coding and represented the total inaccurate claims was calculated by the formula given below:\n\nTotal Financial Impact = Charges before the re-coding process - Total charges of all cases after the recoding process\n\nThe chi-square test was used to assess an association between errors during the coding process and the diagnosis made. Cohen’s Kappa test was used to examine the degree of agreement between the assignment of every code using DRG (https://sourceforge.net/projects/drg-project/) before (by the hospital coders) and after the recoding process (by Health Information Management department staff). Below are mentioned the Kappa values and their interpretations:\n\n• 0.01-0.20 = Low agreement\n\n• 0.21-0.40 = Reasonable agreement\n\n• 0.41-0.60 = Moderate agreement\n\n• 0.61-0.80 = Considerable agreement\n\n• 0.81-1.00 = Excellent agreement\n\nAll data analyses were done using IBM SPSS version 20.0 (https://www.ibm.com/support/pages/spss-statistics-260-fix-pack-1), and a p-value <0.05 was deemed significant.\n\n\nResults\n\nInaccurate coding of primary diagnosis was observed in 57 (26.8 per cent) records, and secondary diagnosis was observed in 21 (9.9 per cent) records (Figure 1).\n\nIncorrect medical coding was most commonly seen in emergency departments, operating rooms, and obstetrics and gynaecology facilities. The Emergency Room had the greatest incidence of incorrect coding at 7.5 per cent, followed by the Surgery Clinic at 4.7 per cent and the Gynecology and Obstetrics Clinic at 2.3 per cent (Table 1). Six (2.8 per cent) incorrect secondary diagnostic documents were found in the examining clinic, four (1.9 per cent) in the nephrology department, and two (1.1 per cent) in the diabetes department. There was only modest agreement between the clinician coder and the staff coder on the primary diagnosis, and only low agreement on the secondary diagnosis with a kappa score of 0.12 (kappa score of 0.48).\n\nFinancial claims before and after re-coding are shown in Figure 2. Because of financial differences caused by inaccurate medical codes for primary and secondary illnesses, certain financial claims have been denied by insurance companies. Before the re-coding process of this research, a total of 42,333 SR (11287.46 $) was in the cash claims, the re-coding procedure was reduced to 29,406 SR (7840.67 $) in total in terms of proper financial claims, incorrect medical coding caused an error of 12,927 SR (3446.79 $) in payment claims.\n\n\nDiscussion\n\nThe process of assigning ICD codes is complicated due to the numerous steps and the participants involved in the process leading to the creation of opportunities for error. Evaluating the accuracy of the code will subsequently improve healthcare decisions.6 Clinical codes are considered wrong or inaccurate when the clinical coder’s codes differ from the independent coding auditor’s code.7 A wrong code for the disease or procedure leads to assigning a wrong DRG code, which eventually will generate an incorrect amount.8 The issue of inaccurate medical coding as observed in the present study and its financial ramifications, not only impacted hospitals’ financial claims but also had a profound effect on the treatment procedure of the patients.9\n\nThe present study reported approximately one-third (36.7 per cent) of the samples were incorrectly recorded in primary and secondary diagnosis, which resulted in the hospital being liable for 12,927 Saudi Riyals (3446.79 $) in terms of lost revenue. The findings found concordance with studies conducted by Zafirah et al.,1 and Jordan et al.4 in the service of liaison psychiatry, and plastic surgery cases. According to the findings of Jordan et al.,4 12.7 per cent of patients switched to a higher-paying DRG and the hospital gained €305,349 in reimbursement. Hospitals might lose up to £29,000 if patients are categorized incorrectly, according to Mole et al.5\n\nOne of the reasons for the commission of errors by the coders is their being of non-medical background. The hospital coders rely on accurate documentation in patient notes, including admission documents, operating notes, and discharge summaries. Hospital coders cannot retrieve information from other sources such as old notes and letters for important information such as medical comorbidities and medications. Therefore, complete and accurate medical clerking by the admitting team is essential for hospital coders to accurately record comorbidities required for HRG tariffs and to reduce errors.9\n\nDuring the present study, various problems were encountered which created hurdles in the process of sample collection. The COVID-19 pandemic restricted access to researchers. Hybrid health records could not gather information electronically and the process of data collection and processing was time-consuming.10 The study failed to have access to procedure codes and documentation during sample collection because of confidentiality issues. Furthermore, private hospitals are also reluctant to produce their financial statements and may attempt to conceal data for fear of exposing financial discrepancies claimed by patients or insurance companies due to their faults.\n\nBefore conducting this research, the researcher obtained the approval from Institutional Review Board of Prince Sultan Military College of Health Sciences (IRB-2021-HIM-023) on June 6, 2023. The study was conducted in accordance with the declaration of Helsinki.\n\nThe consent from the participants was waived by the ethical approval committee.", "appendix": "Data availability\n\nFigShare Research Samples Impact of Inaccurate Clinical Coding on Financial Outcome A Study in a local hospital in Najran, Saudi Arabia.xlsx. [https://doi.org/10.6084/m9.figshare.25908010.v2].\n\nThis project contains the following underlying data:\n\n1. Data file\n\n\nAcknowledgements\n\nThe author is very thankful to all the associated personnel in any reference that contributed to/for this research.\n\n\nReferences\n\nZafirah SA, Nur AM, Puteh SEW, et al.: Potential loss of revenue due to errors in clinical coding during the implementation of the Malaysia diagnosis related group (MY-DRG®) Casemix system in a teaching hospital in Malaysia. BMC Health Serv. Res. 2018; 18: 11–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNouraei SAR, Hudovsky A, Frampton AE, et al.: A study of clinical coding accuracy in surgery: implications for the use of administrative big data for outcomes management. Ann. Surg. 2015; 261(6): 1096–1107. PubMed Abstract \| Publisher Full Text\n\nPreyra C: Coding response to a case-mix measurement system based on multiple diagnoses. Health Serv. Res. 2004; 39(4p1): 1027–1046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJordan I, Barry H, Clancy M, et al.: Financial impact of accurate discharge coding in a liaison psychiatry service. J. Psychosom. Res. 2012; 73(6): 476–478. PubMed Abstract \| Publisher Full Text\n\nMole RJ, Wong KY, Khan M: Accuracy of clinical coding in plastic surgery trauma. Bull. R. Coll. Surg. Engl. 2018; 100(6): 273–276. Publisher Full Text\n\nKhurram SA, Warner C, Henry AM, et al.: Accuracy of clinical coding for procedures in oral and maxillofacial surgery. Br. J. Oral Maxillofac. Surg. 2016; 54(8): 894–897. PubMed Abstract \| Publisher Full Text\n\nCampbell SE, Campbell MK, Grimshaw JM, et al.: A systematic review of discharge coding accuracy. J. Public Health. 2001; 23(3): 205–211. PubMed Abstract \| Publisher Full Text\n\nPongpirul K, Walker DG, Rahman H, et al.: DRG coding practice: a nationwide hospital survey in Thailand. BMC Health Serv. Res. 2011; 11: 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToner E, Khaled A, Ramesh A, et al.: Financial impact of inaccurate coding plus cost-effectiveness analysis for surgically managed patients with periprosthetic fractures. Cureus. 2021; 13(2): e13060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeywood NA, Gill MD, Charlwood N, et al.: Improving accuracy of clinical coding in surgery: collaboration is key. J. Surg. Res. 2016; 204(2): 490–495. PubMed Abstract \| Publisher Full Text" }	[ { "id": "310558", "date": "07 Aug 2024", "name": "Shankar Srinivasan", "expertise": [ "Reviewer Expertise Health Informatics", "hospitalization outcomes research", "health data analytics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is very timely and impactful since it is appropriate to evaluate the current coding practices, their limitations and the costs they have on both patients and hospitals.\n\nThe author have used the appropriate data sources as also methodology for assessing their measures they had set out as being appropriate for the goals of the project. They have also employed the appropriate statistical techniques (fairly straightforward ones given the nature of the project and its goals and which yielded appropriate results for an effective interpretation and reporting. Though the authors have used appropriate data for their analyses it would be great to further increase the data, perhaps from several more hospitals, so that the results may reveal some commonality amongst the hospital departments generating such errors. This will help towards selecting appropriate remedial measures to minimize such occurrences in future. Overall it is a novel project with lot of potential for further research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "12179", "date": "08 Aug 2024", "name": "Salem Albagmi", "role": "Author Response", "response": "Thank you for your valuable insights on data, methodology, and statistical techniques. Your suggestion to expand the dataset is noted for a broader perspective. Understanding common trends can enhance remedial measures. Acknowledging the study's potential for further research is motivating. Continued exploration may lead to significant insights and practical solutions. Your feedback guides the study's future direction for impactful advancements in healthcare coding practices. Thank you for your insightful recommendations." } ] }, { "id": "314504", "date": "22 Aug 2024", "name": "Muteb Alshammari", "expertise": [ "Reviewer Expertise biomedical informatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 effectively addresses the importance of accurate clinical coding and its impact on hospital revenue. The methodology section is well-detailed, explaining the study design and data analysis methods comprehensively. Results are presented logically with figures and tables supporting the findings. The discussion elaborates on the implications of inaccurate coding well, emphasizing the financial consequences and patient care.\nThe manuscript provides valuable insights into the impact of inaccurate clinical coding on hospital finances. It has the potential to make a significant contribution to the field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "12288", "date": "24 Aug 2024", "name": "Salem Albagmi", "role": "Author Response", "response": "Thank you for your valuable feedback" } ] } ]	1	https://f1000research.com/articles/13-820
https://f1000research.com/articles/13-583/v1	05 Jun 24	{ "type": "Brief Report", "title": "Genome assembly catalog for species in the Japanese Red List: unlocking endangered biodiversity through genomic inventory", "authors": [ "Kirill Kryukov", "Naoyuki Nakahama", "Shigehiro Kuraku", "Naoyuki Nakahama" ], "abstract": "Improvements in DNA sequencing technology are allowing the dramatic increase of whole genome data for a wide variety of species. Such genome sequence data can assist the monitoring of intraspecific genetic diversity, but is often lacking for threatened species. In this project, we focused on the national Red List, a catalog of extinct and threatened species, issued by the Japanese government. We combined the data included in it with the record of genome assembly in NCBI and tabulated the assembly availability of the species in the list. The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List as well as a strong bias towards mammals and birds in Animalia and vascular plants in Plantae. Our data presentation highlights potential systematic limitations in genome sequencing (e.g., budget for sequencing large genomes of amphibians) and instructs future policies including which taxon needs more effort for genome sequencing. The resultant tables are available in the original website https://treethinkers.nig.ac.jp/redlist/ and are regularly updated.", "keywords": [ "Biodiversity", "Whole Genome Assembly", "Red List", "Japanese Biota", "Japanese Fauna" ], "content": "Introduction\n\nGenome DNA sequences do not only instruct cellular and other phenomena through genetic readout but also contain the valuable information of genetic diversity when they are analyzed on a population level. Investigation of genetic diversity serves as an irreplaceable navigator of conservation biology (Theissinger et al., 2023), which has been facilitated by recent development of ‘reduced representation sequencing’ methods targeting individuals (Luikart et al., 2018). However, its feasibility largely relies on the availability of whole genome sequences to be used as ‘reference’ sequences.\n\nWhole genome sequences of endangered species have various advantages in conducting conservation studies on endangered species. First, whole genome information can greatly contribute to the use of genetic information from historical specimens. The use of molecular information from past museum specimens, which is known as “Museomics”, has attracted much attention (Fong et al. 2023). In recent years, there have been more studies using museum specimens for conservation genomics (Nakahama 2021). Therein, whole-genome resequencing is a powerful tool. Second, genetic load is quantified from genome information. It is currently difficult to elucidate the mechanism of inbreeding depression itself, as the function and expression mechanism of each deleterious gene is largely unknown. However, it is possible to quantify the genetic load by estimating the amount of deleterious mutations from annotated whole-genome information or transcriptome data (Hamabata et al. 2019; Dussex et al. 2021; Tian et al. 2022; Yoshida et al., 2020). Genomic information also contributes to recent high-resolution estimation of population demography (reviewed in Nadachowska-Brzyska et al. 2022). Population demography is expected to contribute significantly to the understanding of the natural history of endangered species, as well as to the establishment of conservation units and the determination of conservation policy.\n\nThe prevalence of massively parallel sequencing technologies has enabled the acquisition of whole genome sequence information for diverse species. This type of effort has further been accelerated by world-wide trends of biodiversity genomics led by Earth BioGenome Project (EBP) (Gupta 2022; Lewin et al., 2022), and some projects under the EBP are dedicated to promote this trend in particular districts of the world (e.g., Shaffer et al., 2022). Even though whole genomes have been sequenced for a number of species, some of them remain as contigs that have not undergone further steps to build up longer DNA sequences towards a chromosome scale. Prioritization of our effort in conservation biology should be preceded by the identification of ‘cold spots’ based on the listing of potential species requiring conservation effort and monitoring the current status of whole genome sequencing for those species.\n\nJapan has unique fauna and flora and is selected as a “biodiversity hotspot”, but biodiversity in Japan is experiencing rapid declines (Marchese 2015; Kobayashi et al. 2019). In 2020, the Ministry of the Environment of Japan (2020) issued an updated version of the Japanese Red List, which included 5,748 taxa, of which 3,716 are categorized as Critically Endangered (CR), Endangered (EN), and Vulnerable (VU). For some of these species, particularly those at high risk of extinction, whole genome sequences have been determined, and conservation genetic studies have accumulated (Nakahama et al. 2022). To facilitate conservation research on endangered species using genome sequences, it is crucial to monitor the current status and tendency of whole genome sequencing for different taxa to prioritize future policies.\n\nRecognizing the importance of whole genome data and the urgency of the ongoing biodiversity crisis, we decided to start monitoring the availability of assembled genome data for all species in the Japanese Red List. Together with genome data, we also monitor the presence of Red List species in major taxonomic databases. In this paper we describe the structure and methods we use for maintaining our regularly updated resource, which is available online at https://treethinkers.nig.ac.jp/redlist/.\n\n\nMethods\n\nWe use the Japanese Red List, 2020 edition, published by the Ministry of the Environment, Japan. We use the digital copy of the Red List provided by the IKIMONO LOG (https://ikilog.biodic.go.jp/). The Red List data is divided in 13 separate csv files, downloadable at https://ikilog.biodic.go.jp/Rdb/booklist.\n\nThe NCBI Taxonomy database was obtained from the NCBI server at https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/. For each update, we automatically download the latest dump of the NCBI Taxonomy database. For the GBIF database, we downloaded the latest version of the “GBIF Backbone Taxonomy”, released on August 28, 2023, from the GBIF Datasets page (https://www.gbif.org/dataset/ => GBIF Backbone Taxonomy => Download). For the iNat taxonomy, we automatically download the latest dataset “iNaturalist Taxonomy DarwinCore Archive” from https://www.inaturalist.org/pages/developers during each update.\n\nFor the NCBI genome assembly information, we use the NCBI Datasets command line tool (https://www.ncbi.nlm.nih.gov/datasets/docs/v2/download-and-install/) to download genome summaries for all taxa related at up to the family level to Red List entries. The command we use is “datasets summary genome taxon ID”. We automatically download the latest summaries during each update.\n\nThe original csv files of the Red List used two different text encodings: 10 files used the UTF-8 encoding while the other 3 files used the SHIFT-JIS encoding (redlist2020_kairui.csv, redlist2020_invertebrate.csv, and redlist2020_sorui.csv). We converted the SHIFT-JIS-encoded csv files to UTF-8. We share our set of preprocessed csv files at: https://biokirr.com/Japanese-Red-List-Genomes/Red-List-2020-csv-UTF-8.zip.\n\nWe use all unique species or subspecies names from these files. Some entries with the LP (Threatened Local Population) conservation status are listed multiple times, namely for each endangered population. We use only one copy of each of such entries.\n\nWhen we look up the Red List entries in the three taxonomic databases, we first use the entire scientific name of each entry, and try to locate an identically named entry in the taxonomic database. If we can’t find a corresponding taxonomic record, we use synonyms registered in the database (only with the NCBI Taxonomy database). If we still cannot find the taxonomic record, we use our own list of synonyms. Our list of synonyms is shared at: https://biokirr.com/Japanese-Red-List-Genomes/synonyms.txt.\n\nWith the NCBI taxonomy database, we additionally look up the species name and genus name for the entries that can’t be located using the complete scientific name.\n\nMost of our data processing is automated, implemented in a Perl script, freely available at https://biokirr.com/Japanese-Red-List-Genomes/Japanese-Red-List-Genomes-processing-script.zip. This includes downloading and decompressing NCBI genome summaries, downloading and decompressing NCBI and iNat taxonomies, parsing the Red List csv files, and generating all our tables and web pages. We use the uPlot JavaScript library (https://github.com/leeoniya/uPlot) for the timeline chart. We manually initiate the update process periodically, usually at least once a month, and upload the newly generated files to the web server. Our script is available at https://biokirr.com/Japanese-Red-List-Genomes/Japanese-Red-List-Genomes-processing-script.zip. The script is shared under the zlib/libpng license and is free to use, modify and distribute.\n\n\nResults\n\nAll our data was produced by cross-referencing the Japanese Red List (latest edition 2020 as of March 2024) with the major taxonomic and genomic databases. We obtained taxonomic and genomic data from NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/), and additional taxonomic data from GBIF (Global Biodiversity Information Facility, https://www.gbif.org/) and iNat (iNaturalist, https://www.inaturalist.org/). Our results are available online without restrictions at the following url: https://treethinkers.nig.ac.jp/redlist/. The main page includes summary statistics, and links to more detailed tables. We update the website periodically, to reflect the latest taxonomy and genome information. The date of the latest update is shown at the top of the main page.\n\nRegistering whole genome sequences in public database should be preceded by specifying the organism from which the sequences were derived, which is a typical format of the database. The two main data types presented in our newly established resource are the availability of the Red List entries in taxonomic databases, and the availability of whole genome sequences of those Red List entries. The first table at the main page, “Summary by conservation status” (Figure 1), shows the total numbers for each conservation status. The next table, “Summary by taxonomic group” (Figure 2), shows the numbers for the 13 taxonomic groups defined in the Red List. These tables are convenient for seeing how many Red List entries are registered in at least one of the taxonomic databases (either NCBI, GBIF, or iNat), and how many Red List entries already have assembled genomes.\n\nData is shown as of May 2024.\n\nData is shown as of May 2024.\n\nIn the following table, “Comparison of taxonomies”, the three taxonomic datasets that we use are compared side by side with each other, in terms of covering the 13 sections of the Red List. The rightmost column again displays the total number of entries that are registered in any of the three taxonomic databases. The next table, “Taxonomy coverage”, shows how many Red List entries have corresponding NCBI Taxonomy nodes of three different ranks: complete name (can be species, or subspecies or a variety), species, and genus.\n\nRow names (in the first column) in all these summary tables link to detail tables, where taxonomic and genomic information is shown for each individual Red List entry (Figure 3). All detail tables contain one Red List entry per row, and are structured in the same way. First, the conservation status and taxon names (scientific and Japanese names) are shown. The names are shown exactly as provided in the Red List, including names that are misformatted or contain typos, to make it easier to relate our data with the Red List. The next three columns show whether the entry is registered in the taxonomic databases (NCBI, GBIF, and iNat). If a Red List entry is found in the taxonomic database, its table cell contains a check mark, linked to the corresponding entry in the taxonomic database. The check mark color indicates the method of locating the name in taxonomy. Blue check mark means that the exact scientific name used in the Red List is registered in the taxonomic database. Brown or red check mark means that the Red List entry is registered in the taxonomic database under a different name, and that a synonym was used to find the connection. In such cases the check mark color indicates the source of the synonym: brown check mark means that the synonym is registered in the taxonomic database, red check mark means that the synonym is from our own manually constructed dataset of synonyms.\n\nData is shown as of May 2024.\n\nThe “Sequenced? (assembly level)” column shows whether an entry has sequence genome, and the level of the best available assembly (complete > chromosome > scaffold > contig). It also links to the genome assembly pages on the NCBI website. The three columns under the “Number of sequenced genomes” title show the total number of available genomes for organisms in the same species, genus, and family, with the corresponding Red List entry. The next two columns under the “Number of species with sequenced genomes” title show how many distinct species already have sequenced genomes in the same genus and family with the Red List entry. Finally, the last column shows the release date of the earliest available genome assembly for each entry.\n\nThe chart “Changes of sequenced genome availability over time” (Figure 4) on the main page shows how many Red List entries already had genome sequence data at each point of time. It can be seen that genome sequencing accelerated around the beginning of the year 2019, and is currently continuing with the speed of about 40 newly sequenced entries per year.\n\nData is shown as of May 2024.\n\nThe next section on the main page shows the 20 most recently released genomes. Finally, the link “Submitters of sequenced genomes” brings us to the list of organizations that performed genome sequencing, ranked by the number of Red List entries covered. As of May 2024, the Japanese National Institute for Environmental Studies leads the list, with 15 sequenced species (divided into multiple lines because of spelling differences in the registered organization name).\n\n\nDiscussion\n\nWe cross-referenced the Red List with the three major international taxonomic databases: NCBI, GBIF and iNat. NCBI Taxonomy is used for annotating sequence data, and links to all available sequence data in other NCBI databases. GBIF provides literature references and occurrence locations. iNat provides occurrence locations and photographs. These databases are curated independently from each other, and provide different unique data. Together, these three taxonomic resources provide a comprehensive coverage of the worldwide biological diversity. We found that 91.2% of the Red List entries are registered in at least one taxonomic database. It is crucial that 8.9% of Red List entries are not registered in any of the three taxonomic databases that we used. Taxonomic classification is the backbone of biological research, as it enables systematic ways of discussing and describing the relationships between various organisms. Thus, it is expected that all endangered organisms of the Japanese Red List are registered in all major international taxonomic databases.\n\nThe web site generated in this project offers a gateway to monitor the availability of whole genome sequence information for (sub) species in the Japanese Red List. As of May 2024, 2.3% of all the entries (species or subspecies) have whole genome assemblies in NCBI. Mammals and birds are relatively better covered groups, with 21.3% and 19.5% of the species’ genomes already sequenced, respectively. Reptiles and fishes have 9.7% and 8.1% of the entries sequenced. Also, fungi have 8.2% of entries sequenced. The rest of the groups are almost completely uncovered by genome data. For example, the taxon Amphibia in our list contains quite a few salamanders endemic to Japan, and their genomes are exceptionally large, exceeding 10 Gb. The technical difficulty is reflected in the unavailability of genome assemblies in this taxon. In total, only 134 Red List entries have assembled genomes, or about 2.3% of all entries. We expect that the rate of genome sequencing will largely increase, and that eventually assembled genomes will cover the entire Red List.\n\nWhole genome data is increasingly important in biological and conservation research, as it can provide a better understanding of endangered organisms, and help in the conservation efforts. Our comprehensive catalog of genomic and taxonomic information for the Japanese Red List will not only be useful for locating genome assemblies, but, importantly, it will also help focusing the future research efforts and efficiently allocating the scarce resources available for genome sequencing projects. We will continue monitoring the available data and updating our website, and similar efforts are anticipated in other regions of the world, in order to fuel preemptive, evidence-based biodiversity conservation.\n\n\nEthics and consent\n\nNo personal or otherwise human-related data was used in this study. All data we used is already open and public. Therefore, no ethics-related or consent-related issues are applicable to this study.", "appendix": "Data availability\n\nAll our data is placed in the public domain, and shared on the website https://treethinkers.nig.ac.jp/redlist/.\n\n\nAcknowledgments\n\nWe thank Rumiko Suzuki for helpful comments and discussion.\n\n\nReferences\n\nDussex N, van der Valk T , Morales HE, et al.: Population genomics of the critically endangered kākāpō. Cell genomics. 2021; 1(1): 100002. Publisher Full Text\n\nFong JJ, Blom MP, Aowphol A, et al.: Recent advances in museomics: revolutionizing biodiversity research. Front. Ecol. Evol. 2023; 11: 349. Publisher Full Text\n\nGupta PK: Earth Biogenome Project: present status and future plans. Trends in genetics: TIG. 2022; 38(8): 811–820. PubMed Abstract \| Publisher Full Text\n\nHamabata T, Kinoshita G, Kurita K, et al.: Endangered island endemic plants have vulnerable genomes. Communications biology. 2019; 2: 244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobayashi Y, Okada KI, Mori AS: Reconsidering biodiversity hotspots based on the rate of historical land-use change. Biol. Conserv. 2019; 233: 268–275. Publisher Full Text\n\nLewin HA, Richards S, Lieberman Aiden E, et al.: The Earth BioGenome Project 2020: Starting the clock. Proc. Natl. Acad. Sci. USA. 2022; 119(4): e2115635118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarchese C: Biodiversity hotspots: A shortcut for a more complicated concept. Global Ecology and Conservation. 2015; 3: 297–309. Publisher Full Text\n\nMinistry of the Environment, Government of Japan: The Red List of Japan.2020 [cited 8 June 2023]. (In Japanese). Reference Source\n\nLuikart G, Kardos M, Hand BK, et al.: Population genomics: advancing understanding of nature.Rajora O, editor. Population genomics. Cham (Switzerland): Springer; 2018. Publisher Full Text\n\nNadachowska-Brzyska K, Konczal M, Babik W: Navigating the temporal continuum of effective population size. Methods Ecol. Evol. 2022; 13(1): 22–41. Publisher Full Text\n\nNakahama N: Museum specimens: An overlooked and valuable material for conservation genetics. Ecol. Res. 2021; 36(1): 13–23. Publisher Full Text\n\nNakahama N, Ando H, Yoshikawa N, et al.: Genetic information as a basis for conservation genetics of national endangered species of wild fauna and flora in Japan. Japanese Journal of. Conserv. Ecol. 2022; 27: 2128. (in Japanese with English summary). Publisher Full Text\n\nShaffer HB, Toffelmier E, Corbett-Detig RB, et al.: Landscape Genomics to Enable Conservation Actions: The California Conservation Genomics Project. J. Hered. 2022; 113(6): 577–588. PubMed Abstract \| Publisher Full Text\n\nTheissinger K, Fernandes C, Formenti G, et al.: How genomics can help biodiversity conservation. Trends in genetics: TIG. 2023; 39(7): 545–559. Publisher Full Text\n\nTian D, Patton AH, Turner BJ, et al.: Severe inbreeding, increased mutation load and gene loss-of-function in the critically endangered Devils Hole pupfish. Proc. Biol. Sci. 2022; 289(1986): 20221561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshida K, Ravinet M, Makino T, et al.: Accumulation of deleterious mutations in landlocked threespine stickleback populations. Genome Biol. Evol. 2020; 12: 479–492. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "288051", "date": "20 Jun 2024", "name": "James Fleming", "expertise": [ "Reviewer Expertise Biodiversity Genomics", "Ecdysozoa", "Phylogenetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor Comments: The authors present a new resource for the scientific community - a website that tracks the progress of the sequencing of Japanese Red List species. In addition, they analyse the distribution of sequence availability for these species at the time of the resource's foundation. Overall, I believe that https://treethinkers.nig.ac.jp/redlist/ is a fascinating, worthwhile and useful tool for the scientific community, and appreciate efforts to link biodiversity genomics to conservation biology, particularly considering the precarious nature of many ecosystems in the present day. The manuscript itself is well-written, and the analysis of existing Japanese Red List data is thorough and eye-opening. I also appreciate the authors acknowledging the wide variety of scripts employed throughout the project, especially in the design and presentation of the figures.\nHowever, I do think that there are two important areas which go unaddressed in the manuscript in its current form. 1) The focus on the Japanese Red List is useful for defining the scope of the project, but Japan maintains two Red Lists concurrently: one for terrestrial and freshwater organisms, and one for marine. Although it would be an extension of the scope of the manuscript as presented, expanding the tool to include the Marine Red List would be a huge boon to the proposal, especially considering the size of Japan's coastline, and the diversity of life inside it.\n2) More conceptually, I have reservations about the use of the Red List to direct genome sequencing efforts in the near future, as the authors propose. The manuscript feels like it is missing a section in both its introduction and discussion where it considers the flaws of Red List methodologies, and the limitations that they might present to the tool. The Red List is very heavily biased towards larger organisms, and particularly vertebrates, meaning that the existing mammal and bird bias already noted by the authors in the results section of the manuscript is in actuality a bias within a bias. My concern is that directing policy towards \"completing the Red List\" might actually result in more species falling through the cracks, particularly in areas of biology that are already chronically underfunded. There is a strong body of literature on this subject, and it would greatly improve the manuscript to address it - I've added some useful references below that might help as a starting point: Possingham H.et.al.,2002 (ref 1) Cazalis V et.al., 2022 (Ref 2) Régnier C, et.al., 2009 (ref 3) Goodsell R et.al., 2024 (ref 4)\nThat said, I do believe that this tool is a potentially incredibly useful resource for researchers of Red List species, and I appreciate efforts to unite the two disciplines towards their closely connected goals.\nVery Minor Comments: Abstract: \"are allowing the dramatic increase of whole genome data for a wide variety of species.\" -> \"are allowing for the dramatic increase of whole genome data for a wide variety of species.\" \"genome sequence data can assist the monitoring of intraspecific genetic diversity\" -> \"genome sequence data can assist in the monitoring of intraspecific genetic diversity\" \"The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List\" -> \"The combined data shows that whole genome sequence data is available for a low percentage (2.1%) of the taxa ranked on the Japanese Red List\" \"instructs future policies including which taxon needs more effort for genome sequencing.\" -> \"instructs future policies, including which taxa require greater effort in genome sequencing.\"\nIntroduction: \"Genome DNA sequences do not only instruct cellular and other phenomena through genetic readout but also contain the valuable information of genetic diversity when they are analyzed on a population level.\" -> \" but also contain valuable information about genetic diversity when they are analyzed on a population level.\" \"some projects under the EBP are dedicated to promote this trend\" -> \" some projects under the EBP are dedicated to promoting this trend\"\nDiscussion: \" It is crucial that 8.9% of Red List entries are not registered in any of the three taxonomic databases that we used.\" - I wasn't quite sure what this sentence was trying to be highlight, so it might be useful to rephrase it. \" the taxon Amphibia in our list contains quite a few salamanders\" -> how many? I think a number could really help support the overall thrust of the point. \" The technical difficulty is reflected in the unavailability of genome assemblies in this taxon.\" -> \" The technical difficulty is reflected in the lack of available genome assemblies for this taxon.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "12005", "date": "22 Jul 2024", "name": "Kirill Kryukov", "role": "Author Response", "response": "We sincerely appreciate the encouraging comments and discussion. We have modified the website and revised the manuscript. Point-by-point replies: Reviewer Comment: 1) The focus on the Japanese Red List is useful for defining the scope of the project, but Japan maintains two Red Lists concurrently: one for terrestrial and freshwater organisms, and one for marine. Although it would be an extension of the scope of the manuscript as presented, expanding the tool to include the Marine Red List would be a huge boon to the proposal, especially considering the size of Japan's coastline, and the diversity of life inside it. Author Response: - Thank you for the nice suggestion. We now incorporated the Marine Red List data to our analysis, and we agree that the resulting resource is more complete and interesting. Reviewer Comment: 2) More conceptually, I have reservations about the use of the Red List to direct genome sequencing efforts in the near future, as the authors propose. The manuscript feels like it is missing a section in both its introduction and discussion where it considers the flaws of Red List methodologies, and the limitations that they might present to the tool. The Red List is very heavily biased towards larger organisms, and particularly vertebrates, meaning that the existing mammal and bird bias already noted by the authors in the results section of the manuscript is in actuality a bias within a bias. My concern is that directing policy towards \"completing the Red List\" might actually result in more species falling through the cracks, particularly in areas of biology that are already chronically underfunded. There is a strong body of literature on this subject, and it would greatly improve the manuscript to address it - I've added some useful references below that might help as a starting point: Possingham H.et.al.,2002 (ref 1) Cazalis V et.al., 2022 (Ref 2) Régnier C, et.al., 2009 (ref 3) Goodsell R et.al., 2024 (ref 4) Author Response: - Thank you very much for your suggestion. We have modified the relevant parts to draw readers' attention to criticisms of biased species selection. Reviewer Comment: Very Minor Comments: .......genome assemblies for this taxon.\" Author Response to Reviewer's Minor Comments: - We appreciate all minor comments, and we addressed them in the revised manuscript." } ] }, { "id": "292685", "date": "25 Jun 2024", "name": "Guilherme Borba Neumann", "expertise": [ "Reviewer Expertise Bioinformatics", "Conservation 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 current work proposes a simple way of summarizing current numbers and efforts on available whole-genome sequence information for endangered Japanese species. Although simple, the developed website provides a clear picture of needed efforts on the sequencing of most endangered species in Japan, generating a base for further studies and ultimate impact on the conservation of endangered species. Nevertheless, I suggest a few changes in the manuscript: In the introduction I would add some information about the total number of species in Japan, and also endemic Japanese species in order to compare with the number of species in the red list. It would be also interesting to know from the current available assemblies, which are annotated and presented as a Reference Genome, rather than just an assembly. Further information about the quality of the assemblies, such as number of contigs, N50 etc would also be interesting to have. Have the authors considered ways of automating the updates ? So that this nice work does not stop here, but guarantees a continuous monitoring of the development of genomics in endangered species. Other databases were left out, such as ensembl (https://rapid.ensembl.org/index.html), which now has a rapid version of publishing new assemblies, and GOAT (https://goat.genomehubs.org/). Actually the work here described is very similar to what has been done on GOAT. Therefore, I would suggest adding some discussion on similar efforts, maybe even comparing how well or bad represented are the Japanese species in those projects. Finally, I would rather add the actual figures in the manuscript than screenshots. The quality of screenshots are inferior, and it does not look very professional. In addition, it is important to add the meaning of the abbreviations in the figures as part of the figure description/legends as well.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "12006", "date": "22 Jul 2024", "name": "Kirill Kryukov", "role": "Author Response", "response": "We sincerely appreciate the kind comments and suggestions. We have modified the website and revised the manuscript. Point-by-point replies: Reviewer Comment: 1. In the introduction I would add some information about the total number of species in Japan, and also endemic Japanese species in order to compare with the number of species in the red list. Author Response: - Thank you for the suggestion. We added the number of taxa evaluated for the Red Lists, in the Introduction. As for the total number of species in Japan, there exists an estimate of about 90,000 species, but it is based on literature in the 1990s and is not up-to-date, so we decided to not include this less reliable number. Reviewer Comment: 2. It would be also interesting to know from the current available assemblies, which are annotated and presented as a Reference Genome, rather than just an assembly. Further information about the quality of the assemblies, such as number of contigs, N50 etc would also be interesting to have. Author Response: - Thank you for the suggestion. We added this data to the tables. We also note that other information about each genome assembly can be found easily, since the data in our tables also serves as a link to the corresponding genome in the NCBI Datasets. Reviewer Comment: 3. Have the authors considered ways of automating the updates ? So that this nice work does not stop here, but guarantees a continuous monitoring of the development of genomics in endangered species. Author Response: - Thank you for the suggestion. Indeed, we already automated the updates, and we are performing them periodically, usually about once a month. Reviewer Comment: 4. Other databases were left out, such as ensembl (https://rapid.ensembl.org/index.html), which now has a rapid version of publishing new assemblies, and GOAT (https://goat.genomehubs.org/). Actually the work here described is very similar to what has been done on GOAT. Therefore, I would suggest adding some discussion on similar efforts, maybe even comparing how well or bad represented are the Japanese species in those projects. Author Response: - Thank you for the comment. We agree that it could be useful to monitor other genome databases. We focus on GenBank genomes as it is the largest repository of genome data, and genomes released elsewhere tend to get submitted to GenBank eventually. We added a comment about this in a discussion, and we suggest that this could be a topic of improvement in the future. Reviewer Comment: 5. Finally, I would rather add the actual figures in the manuscript than screenshots. The quality of screenshots are inferior, and it does not look very professional. In addition, it is important to add the meaning of the abbreviations in the figures as part of the figure description/legends as well. Author Response: - Thank you for the suggestion. We removed the distracting borders from the figures. Our data is presented via a website, which readers will see with a web browser, therefore screenshots are the best representation of what readers will find when they explore our resource. We added the meaning of all abbreviations to figure legends." } ] } ]	1	https://f1000research.com/articles/13-583
https://f1000research.com/articles/12-1227/v1	27 Sep 23	{ "type": "Study Protocol", "title": "A prospective comparative study of preoperative and postoperative clinicopathological correlation of fibroadenoma of breast: A study protocol", "authors": [ "Sandeep Reddy Ramala", "Suresh R Chandak", "Meenakshi Chandak", "Suresh R Chandak", "Meenakshi Chandak" ], "abstract": "Fibroadenoma of the breast most often arises during the late teens and early reproductive years. It is the most prevalent tumor in women < 30 years. After the age of 40 to 45, fibroadenomas are rarely observed as new masses in women. Fibroadenomas are made up of both epithelial and stromal components. Fibroadenoma is the most frequent type of breast tumor. It is crucial to diagnose preoperatively, both clinically and pathologically, by using F.N.A.C. Postoperatively, Fibroadenoma is confirmed histopathology. However, malignant transformation is very rare in Fibroadenoma of the breast. Early identification, accurate analysis, and appropriate management are advantageous. The current study's goal is to examine preoperative and postoperative clinicopathological aspects of Fibroadenoma and to assess any differences between preoperative and postoperative diagnosis in patients admitted to Jawaharlal Nehru Medical College, Sawangi (M), Wardha, Maharashtra, India.", "keywords": [ "Fibroadenoma", "Breast", "F.N.A.C.", "Histopathological Examination", "Benign" ], "content": "Introduction\n\nFibroadenoma is a common cause of benign breast lumps in young women between the ages of 15 and 35.1 In Fibroadenoma of the breast, clinical evaluation and fine needle aspiration biopsy is the primary component in formulating a diagnosis and predicting benign or malignant breast lump. Cancer in newly found Fibroadenoma is incredibly uncommon.2 Fibroadenomas frequently appear as firm, easily movable masses that wax and wane with the menstrual cycle and may become larger over several months. Fibroadenoma causes no pain, when touched, moves easily within the breast tissue. All patients with breast lumps in the premenopausal period should be suspected of breast fibroadenoma. Fibroadenomas typically have a size of 2 to 3 cm, but they can grow to > 10 cm and result in asymmetry or hypertrophy of the breasts. The eventual treatment plan depends upon the diagnosis of a breast lump. The applicable methods for diagnosing breast lumps are core needle biopsy (C.N.B.) and fine-needle aspiration cytology (F.N.A.C.). Recent years have seen the development of F.N.A.C. and C.N.B. as highly effective diagnostic techniques for examining palpable breast masses.3 Either a core biopsy or a tiny needle aspiration is used for tissue sampling. The simplicity of the procedure, low cost, and, most importantly, low risk of complications are some of the benefits of F.N.A.C. It is minimally invasive, doesn't call for anesthesia, and is generally patient-friendly.4,5 Additionally, two to four days following aspiration, the results are accessible. The gold standard diagnostic test for Fibroadenoma of the breast is considered to be a surgical biopsy pathological examination. Since it is normal for fibroadenomas to diminish naturally, the majority are just watched. Surgery is advised when the Fibroadenoma becomes larger or the symptoms worsen. Recent research has shown that the prevalence of breast disease is rising. After the age of 40, it is seldom ever observed in women. Fibroadenomas are well-encapsulated lumps that can easily separate from the breast tissue around them. Additionally, many women can avoid excision because a sizeable number of fibroadenomas over a 5-year period remain static or shrink in size.6 Sixty-five percent of patients with fibroadenomas that contain cancer have lobular in situ malignant neoplasms.7\n\n\nProtocol\n\nThe aim of this study is to compare Preoperative and Postoperative Clinicopathological Correlation of Fibroadenoma of the Breast.\n\n\n\n1) To thoroughly examine the clinicopathological characteristics of breast fibroadenoma.\n\n2) To compare the clinicopathological aspects of Fibroadenoma before and after surgery.\n\n3) To evaluate the discrepancies between Fine Needle Aspiration Cytology and Histopathological Examination.\n\n4) To Know the Sensitivity and Specificity of Fine needle aspiration cytology.\n\n5) To study different clinical signs elicited in Fibroadenoma.\n\n6) To study the incidence of different benign breast diseases.\n\n\nMethods\n\nEthical approval received by Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha (Approval number DMIHER (DU)/IEC/2023/953).\n\nThe study will be carried out at the Acharya Vinoba Bhave Rural Hospital, a tertiary care facility affiliated with the Jawaharlal Nehru Medical College in Sawangi, Meghe, Wardha, Maharashtra.\n\nIn this prospective comparative study, a total of 75 Patients attending the outpatient department/admitted to the Inpatient department (I.P.D.) of the Surgery department at A.V.B.R.H., Sawangi, Wardha, fulfilling the inclusion criteria will be chosen.\n\n\n\n• All patients admitted to Our Hospital with a provisional clinical diagnosis of Fibroadenoma and willing to give consent for the study.\n\n• Premenopausal women.\n\n\n\n• Postmenopausal women\n\n• Women on hormonal therapy\n\n• Lactating women\n\n• previously operated breast\n\nAfter receiving the written informed consent from study participants to participate in the study and for publication of their data, patients who presented with a history of a breast lump were clinically, and F.N.A.C. was confirmed to have Fibroadenoma of the breast. Patients will be asked about their age, residence, social status, clinical history, past history, personal history, family history and will undergo physical examination of breast, local examination of breast, inspection and palpation of breast, examination of lymph nodes, blood investigations, F.N.A.C. preoperatively and histopathology postoperatively in that order. Frequency and percentage were used to express categorical data. The Chi-square test was used to analyze the relationship between variables. Mean and Standard Deviation were used to express quantitative data. For diagnosis, histopathological findings were linked with F.N.A.C. Calculations were made for the test's sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. Statistics were considered significant for P values under 0.05.\n\nSample size\n\nDaniel formula for sample size:\n\nWhere\n\nZ α/22 is the level of significance at 5 %, i.e., 95 % confidence interval = 1.96\n\nP = Prevalence of fibroadenoma of breast =10./. = 0.10\n\nE = Desired Error of margin = 7% = 0.07\n\nn = 75 patients needed in the study.\n\nStudy reference:\n\nFormula reference: Daniel et al. (1977)\n\nThe results of article will be published in an indexed journal.\n\nThe proposed study is not yet started. The study is expected to begin in July 2023.\n\n\nDiscussion\n\nIt is a prospective comparative study comparing the clinicopathological connection of breast fibroadenoma before and after surgery. Histopathological findings were correlated with F.N.A.C. for the diagnosis of the disease is essential to know how efficient F.N.A.C. is in diagnosing Fibroadenoma of the breast preoperatively.\n\nEarly detection and treatment are crucial to lowering mortality rates in breast cancer cases, and cancer in newly detected Fibroadenoma is exceedingly rare.8 The main usage of imaging techniques is for screening. In the majority of patients, treatment depends upon F.N.A.C. (fine needle aspiration cytology) and C.N.B. (core needle biopsy) reports, which claim to be extremely accurate and closely resemble histological results.3\n\nF.N.A.C. is less invasive than core needle biopsies and is more accurate, affordable, and simple to do.9 The common cause of a false negative cytological diagnosis is known to be the absence of a lesion on sampling during aspiration.10 False-negative and ambiguous diagnoses are notably common in F.N.A.C. of invasive lobular cancer. This is due to the fact that a paucicellular smear with mild atypia and infrequent single intact epithelial cells is more likely to result from classic invasive lobular cancer.11\n\nFeichter et al. found that out of 1472 FNAC of the breast. Cytological diagnosis was benign in 1003 cases(68.1%), 239(16.2%) aspirates were inadequate, false negative cases were (9%), and false positive cases were (0.5%).12\n\nKuijper et al. Conducted a study of histopathological features of Fibroadenoma of the breast in 396 cases. In 32.3% of cases, hyperplasia was discovered. Carcinoma in situ was found in 8 cases (2.0%). In 40.4% of cases, complex histologic characteristics were present, primarily in somewhat older patients. In 8.8% of cases, the surrounding tissue had hyperplasia, which was typically present in older patients.12", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nFibroadenoma of the breast - PubMed: [cited 2023 Jun 6]. Reference Source\n\nPreoperative And Postoperative Clinicopathological Correlation Of Fibroadenoma Of Breast. IJSR - International Journal of Scientific Research (IJSR), IJSR\|World Wide Journals. [cited 2023 Jun 6]. Reference Source\n\nNassar A: Core needle biopsy versus fine needle aspiration biopsy in breast--a historical perspective and opportunities in the modern era. Diagn. Cytopathol. 2011 May; 39(5): 380–388. PubMed Abstract \| Publisher Full Text\n\nKar A, Satapathy B, Pattnaik K, et al.: Trucut Biopsy vs FNAC of Pelvic Tumors-Who Wins the Match? J. Cytol. 2018; 35(3): 179–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoschetta M, Telegrafo M, Carluccio DA, et al.: Comparison between fine needle aspiration cytology (FNAC) and core needle biopsy (CNB) in the diagnosis of breast lesions. G. Chir. 2014 Sep 1; 35(7–8): 171–176. Publisher Full Text\n\nCarty NJ, Carter C, Rubin C, et al.: Management of Fibroadenoma of the breast. Ann. R. Coll. Surg. Engl. 1995 Mar; 77(2): 127–130. PubMed Abstract\n\nPick PW, Iossifides IA: Occurrence of breast carcinoma within a fibroadenoma. A review. Arch. Pathol. Lab. Med. 1984 Jul; 108(7): 590–594. PubMed Abstract\n\nSung H, Ferlay J, Siegel RL, et al.: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021 May; 71(3): 209–249. PubMed Abstract \| Publisher Full Text\n\nGarbar C, Curé H: Fine-needle aspiration cytology can play a role in neoadjuvant chemotherapy in operable breast cancer. ISRN Oncol. 2013; 2013: 1–5. Publisher Full Text\n\nEllis IO, Humphreys S, Michell M, et al.: Best Practice No 179. J. Clin. Pathol. 2004 Sep; 57(9): 897–902. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang S, Ioffe O, Lee I, et al.: Cytologic diagnosis of invasive lobular carcinoma: factors associated with negative and equivocal diagnoses. Diagn. Cytopathol. 2004 Aug; 31(2): 87–93. PubMed Abstract \| Publisher Full Text\n\nKuijper A, Mommers EC, van der Wall E , et al.: Histopathology of fibroadenoma of the breast. Am. J. Clin. Pathol. 2001 May; 115(5): 736–742. Publisher Full Text" }	[ { "id": "261966", "date": "17 Apr 2024", "name": "Nilotpal Chowdhury", "expertise": [ "Reviewer Expertise Cytopathology", "Histopathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. With the presently calculated sample size, the researchers will get only 0.1*75 =7-8 cases of fibroadenoma for their study. Just these many cases of fibroadenoma is not sufficient to fully describe the clinicopathological characteristics of breast fibroadenomas. The sample size formula is appropriate only if the prevalence is the study objective. Increasing the sample size will help in providing relevant answers to the research questions. 2. It is unclear what is meant by clinicopathological aspect of fibroadenoma before and after surgery. After surgery, the fibroadenoma will be removed. Possibly the authors meant something else which may be clarified. 3. This is a cross sectional study, and if consecutive cases are taken, one can study the prevalence and not the incidence of the disease (point 6 of objectives). 4. Reference 9 states that fine needle biopsy is more accurate. This is possibly not true. 5. There is a discrepancy between the sections titled study design (\"a total of 75 Patients attending the outpatient department/admitted to the Inpatient department (I.P.D.) of the Surgery department\") and inclusion criteria (\"All patients admitted to Our Hospital\"). It has to be clarified whether patients attending only OPD will be included. In that case, how will cyto-histological correlation be done?\nEnglish: There is interchange between the tenses used. In some cases , the future tense is used (e.g., \"will be asked\") and in some cases the past tense (\" Chi-square test was used to analyze\") . This needs to be modified accordingly.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "12013", "date": "19 Jul 2024", "name": "Sandeep Reddy Ramala", "role": "Author Response", "response": "1. Sample size - I had calculated sample size as per literature in standardized manner. sample size is calculated by using Daniel formula. In my study I had included total 75 patients. 2. Here we are including patients who are who presented with a history of a breast lump were clinically, and F.N.A.C. was confirmed to have Fibroadenoma of the breast. Patients will be exposed to history taking, clinical examination, and investigations in that order. who presented with a history of a breast lump were clinically, and F.N.A.C. was confirmed to have Fibroadenoma of the breast. post operatively we will compare with histopathological examination. to study in detail clinical and cytological findings and assessment of fibroadenoma of breast. 3. I will change my objective no 6- To study the prevalence of different benign breast diseases. 4. FNAC is more accurate when compare to other investigation and helps in diagnosing in patients with swelling. I think its more accurate. 5. Only I.P.D patients are included in study. Will change it. Thankyou sir for your valuable review. We will do changes accordingly. Thankyou for publishing in your esteemed journal" } ] }, { "id": "261982", "date": "02 May 2024", "name": "Omar Hamdy", "expertise": [ "Reviewer Expertise Surgical oncology", "breast 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\nThanks for your work. The following remarks are to be noted:\n1. Abstract:\nFNAC is not crucial in fibroadenoma in modern surgical practice. Early identification does not make a significant difference in fibroadenoma.\n2. Introduction:\nAgain, FNAC is not an essential part in fibroadenoma management. “After the age of 40, it is seldom ever observed in women. Fibroadenomas are well-encapsulated lumps that can easily separate from the breast tissue around them.” This sentence is repeated. It has been mentioned at the start of the introduction. The last sentence in the introduction does not add to the context.\n3. Study protocol:\nWhy were women with previously operated breast excluded? The criteria to include a tumor and go for surgery is not clear at all. What is the cutoff size to include a patient in the study? In addition, the idea of the study does not add to the already available literature at all.\n4: Discussion: Some parts in the discussion are already repeated in the introduction.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? 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": [ { "c_id": "12014", "date": "19 Jul 2024", "name": "Sandeep Reddy Ramala", "role": "Author Response", "response": "1. Abstract - In most of Indian hospitals. it was standard technique that FNAC is done in most of the breast lump patients. because as number of cases of breast carcinoma is increasing in India. It is better to do FNAC before performing surgery to make tissue diagnosis. because FNAC is low cost, easy procedure, patient can afford in India, simple procedure without more complications. - Early identification does not make difference in fibroadenomas because fibroadenomas are not cancerous 2. Introduction - I will remove repeated sentence. - Last sentence tells that patients with both fibroadenoma and carcinoma in these patients Sixty-five percent contain cancer have lobular in situ malignant neoplasms . 3.Study protocol - Previously operated breast were not excluded. I will rewrite exclusion criteria - The criteria to include a patient to undergo for surgery is patients presented with a history of a breast lump were clinically, and F.N.A.C. was confirmed to have Fibroadenoma of the breast. it can be of any size of lump. - The idea of study is to know Sensitivity and Specificity of Fine needle aspiration cytology. 4. Discussion- repeated parts will be removed Thank you sir for your valuable review. We will do changes as per advised. Thank you for publishing protocol in your esteemed journal" } ] } ]	1	https://f1000research.com/articles/12-1227
https://f1000research.com/articles/13-817/v1	19 Jul 24	{ "type": "Data Note", "title": "A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry", "authors": [ "Michael S. Biddle", "Charles Alende", "Maryam Fotouhi", "Carolyn Jones", "Riham Ayoubi", "Kathleen Southern", "Carl Laflamme", "Harvinder Virk", "NeuroSGC/YCharOS/EDDU collaborative group", "ABIF consortium", "Michael S. Biddle", "Charles Alende", "Maryam Fotouhi", "Carolyn Jones", "Riham Ayoubi", "Kathleen Southern", "Harvinder Virk" ], "abstract": "Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.", "keywords": [ "Uniprot ID P21579", "SYT1", "Synaptotagmin-1", "antibody characterization", "antibody validation", "western blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nNeurons communicate through regulated neurotransmitters release, a process controlled by calcium-dependent exocytosis of synaptic vesicles (SV).1 Synaptotagmin-1, encoded by SYT1, is a transmembrane SV protein, with two tandem C2-domains (C2A and C2B). These domains sense and bind calcium ions, triggering a conformational change that induces SNARE-mediated fusion. Sytnaptotagmin-1 thus couples Ca2+ influx to synchronous SV release of neurotransmitter to the presynaptic cleft.2–5\n\nMutations or dysregulation to SYT1 disrupts synaptic proteins and the synchronous release of neurotransmitters, causing damaging effects to the central nervous system, and contributes to neurodevelopmental conditions including epilepsy,6 autism spectrum disorder7 and neurodegenerative diseases such as Alzheimer’s disease.8 Synaptotagmin-1 is a focal research target, presenting as a potential biomarker for synaptic transmission in neurological health and disease.3,5,9–11\n\nThis research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data.12–14 Here we evaluated the performance of thirteen commercial antibodies for Synaptotagmin-1 for use in western blot, immunoprecipitation, immunofluorescence, and flow cytometry enabling biochemical and cellular assessment of Synaptotagmin-1 properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1).15\n\nThe authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway.16\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from WT (wild type) and KO cells.17,18 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). HCT 116 cell line, which expresses the Synaptotagmin-1 transcript at 4.6 log2 (TPM+1), was identified as a suitable cell line and was modified with CRISPR/Cas9 to KO the corresponding SYT1 gene (Table 1).\n\nFor western blot experiments, WT and SYT1 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with thirteen Synaptotagmin-1 antibodies in parallel (Table 2, Figure 1).\n\n* Monoclonal antibody.\n\n** Recombinant antibody.\n\nLysates of HCT 116 (WT and SYT1 KO) were prepared and 40 μg of protein were processed for western blot with the indicated Synaptotagmin-1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WPX42012BOX) to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 14511-1-AP and 68043-1-Ig* which were titrated as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: A0992 at 1/1000, ARP59447 at 1/1000, MAB4364* at 1/1000, 14558** at 1/1000, mAB 30 (asv30)* at 1/10, mAB 48 (asv8)* 1/10, GTX637119 at 1/1000, GTX637252 1/1000, 14511-1-AP at 1/1000, 68043-1-Ig* 1/10000, 105 008** at 1/1000, 105 011* at 1/1000, MA1-25568* at 1/500. Predicted band size: 47.5 kDa. Monoclonal antibody; Recombinant antibody.\n\nWe then assessed the capability of all thirteen antibodies to capture Synaptotagmin-1 from HCT 116 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific Synaptotagmin-1 antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE (Figure 2).\n\nHCT 116 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated Synaptotagmin-1 antibodies pre-coupled to Dynabeads protein A or protein G. The concentration of 14558* is unknown and therefore 10 μl of this antibody was tested. Samples were washed and processed for western blot with the indicated Synaptotagmin-1 antibody on a precast midi 4-20% Tris-Glycine polyacrylamide gel. For western blot, GTX637119** was used at 1/1000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; HC= antibody heavy chain. Monoclonal antibody; Recombinant antibody.\n\nFor immunofluorescence, thirteen antibodies were screened using a mosaic strategy. First, HCT 116 WT and SYT1 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Synaptotagmin-1 antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested,15 and the images presented in Figure 3 are representative of this analysis.\n\nHCT 116 WT and SYT1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated Synaptotagmin-1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution. The rest of the antibodies were tested at 1 and 2 μg/ml and the final concentration was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilution used: ARP59447 at 1/500, MAB4364 at 1/500, 14558** at 1/50, mAB 30 (asv30)* at 1/500, mAB 48 (asv8)* at 1/30, GTX637119 at 1/500, GTX637252 at 1/250, 14511-1-AP at 1/500, 6803-1-Ig* at 1/1000, 105 008** at 1/1000, 105 011 at 1/500, MA1-25568* at 1/1000. Bars = 10 μm. Monoclonal antibody; Recombinant antibody.\n\nFor flow cytometry, HCT 116 WT and SYT1 KO cells were labelled with distinct fluorescent dyes and combined at a 1:1 ratio. Both cell lines were fixed, permeabilized and blocked in the same tube prior to antibody staining to reduce bias. Nine Synaptotagmin-1 antibodies were then evaluated, with fluorescent intensity assessed using the Attune NxT flow cytometer. Antibody staining in both WT and KO line was then quantified using FlowJo software, with representative histograms presented (Figure 4).\n\nHCT 116 WT and SYT1 KO cells were labelled with a green or violet, fluorescent dye, respectively. WT and KO cells were mixed in a 1:1 ratio, fixed in 4% PFA and permeabilized in 0.1% saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies. Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the KO population (pink histogram, dashed line) compared to the WT cells (green histogram, solid line). Histograms with dotted lines represent secondary antibody-only controls in both WT and KO cells. All primary antibodies were diluted to 1 μg/ml except for 14558* which was used at 0.35 μg/ml (1/100) as the concentration was unknown. Monoclonal antibody; *Recombinant antibody.\n\nIn conclusion, we have screened thirteen Synaptotagmin-1 commercial antibodies by western blot, immunoprecipitation, immunofluorescence and flow cytometry by comparing the signal produced by the antibodies in human HCT 116 WT and SYT1 KO cells. Several high-quality and renewable antibodies that successfully detect Synaptotagmin-1 were identified in all applications. Researchers who wish to study Synaptotagmin-1 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.\n\nThe underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.19\n\n\nMethod\n\nThe standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1).15\n\nCell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID.20,21\n\nHCT 116 WT and SYT1 KO cells were detached, and three million cells were labelled with CellTracker green or violet fluorescent dyes, respectively (Thermo Fisher Scientific, cat. number C7025 and C10094). WT and KO cells were then centrifuged at 300 × g, for 10 min and resuspended in 1% bovine serum albumin (BSA) (Wisent, cat. number 800-095) phosphate-buffered saline (PBS) (Wisent, cat. number 311-010). Cell populations were then combined at a 1:1 ratio, centrifuged and fixed on ice for 20 min using 800 μl of 4% PFA in PBS (Thermo Fisher Scientific, cat. number J61899). Following fixation, 1.2 ml of 1% BSA in PBS was added to the tube, vortexed and centrifuged at 600 × g for 15 min at 4°C. Cells were permeabilized in PBS with 0.1% saponin for 10 min at room temperature, centrifuged at 600 × g for 15 min at 4°C and then blocked with 5% goat serum (Gibco, cat. number 16210-064), 1% BSA in PBS for 30 min on ice. 400,000 cells were aliquoted into individually labelled tubes, centrifuged 600 × g for 15 min at 4°C and incubated in 150 μl of 1% BSA, 0.1% saponin PBS with primary Synaptotagmin-1 antibodies for 30 min on ice. 500 μl of 1% BSA, 0.1% saponin PBS was added to each tube, vortexed and centrifuged at 600 × g for 15 min at 4°C. Cells were then incubated the corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies (0.83 μg/ml) (Proteintech, cat. number RGAR005 and RGAM005) in 150 μl of 1% BSA, 0.1% saponin PBS for 30 min on ice. 500 μl of 1% BSA, 0.1% saponin PBS was added to each tube, vortexed and centrifuged 600 × g 15 min at 4°C.\n\nTubes were resuspended in 1 ml of 1% BSA in PBS and data was acquired using the Attune NxT flow cytometer. Data was analysed using FlowJo with the following gates. The cell population was gated on FSC-A vs SSC-A, within that gate single cells were selected by FSC-A vs FSC-H and then KO and WT cells were isolated by BL1-A vs VL1-A using a quadrat gate. Quantification of antibody staining was then observed in the RL1-A channel and histograms merged to demonstrate the staining intensity between the two populations. The figure was then assembled using Adobe Illustrator 2024.", "appendix": "Data availability\n\nZenodo: Dataset for the Synaptotagmin-1 antibody screening study, https://doi.org/10.5281/zenodo.12636746. 19\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.\n\nABIF consortium: Claire M. Brown and Joel Ryan.\n\nThank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.\n\n\nReferences\n\nBrose N, Petrenko AG, Südhof TC, et al.: Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science. 1992; 256(5059): 1021–1025. Publisher Full Text\n\nTucker WC, Weber T, Chapman ER: Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science. 2004; 304(5669): 435–438. Publisher Full Text\n\nYoshihara M, Littleton JT: Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release. Neuron. 2002; 36(5): 897–908. Publisher Full Text\n\nGeppert M, Goda Y, Hammer RE, et al.: Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 1994; 79(4): 717–727. PubMed Abstract \| Publisher Full Text\n\nCourtney NA, Bao H, Briguglio JS, et al.: Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin. Nat. Commun. 2019; 10(1): 4076. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaitsev А, Amakhin D, Dyomina A, et al.: Synaptic dysfunction in epilepsy. J. Evol. Biochem. Physiol. 2021; 57(3): 542–563. Publisher Full Text\n\nYeo XY, Lim YT, Chae WR, et al.: Alterations of presynaptic proteins in autism spectrum disorder. Front. Mol. Neurosci. 2022; 15: 1062878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÖhrfelt A, Brinkmalm A, Dumurgier J, et al.: The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer’s disease. Alzheimers Res. Ther. 2016; 8(1): 41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChicka MC, Hui E, Liu H, et al.: Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat. Struct. Mol. Biol. 2008; 15(8): 827–835. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Boven MA , Mestroni M, Zwijnenburg PJG, et al.: A de novo missense mutation in synaptotagmin-1 associated with neurodevelopmental disorder desynchronizes neurotransmitter release. Mol. Psychiatry. 2024. Publisher Full Text\n\nRiggs E, Shakkour Z, Anderson CL, et al.: SYT1-Associated Neurodevelopmental Disorder: A Narrative Review. Children (Basel). 2022; 9(10). Publisher Full Text\n\nAyoubi R, Ryan J, Biddle MS, et al.: Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023; 12: 12. Publisher Full Text\n\nCarter AJ, Kraemer O, Zwick M, et al.: Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24(11): 2111–2115. PubMed Abstract \| Publisher Full Text\n\nLicciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13(12): 1446–1459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, Ryan J, Bolivar SG, et al.: A consensus platform for antibody characterization (Version 1). Protocol Exchange. 2024.\n\nBiddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12: 1344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAyoubi R, Laflamme C: Dataset for Synaptotagmin-1 antibody screening study. [Data set]. 2024.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "310140", "date": "19 Aug 2024", "name": "Karl D Murray", "expertise": [ "Reviewer Expertise Neurodevelopment", "plasticity", "neurological disorders", "antibody tool development and characterization" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Biddle et al., present the characterization of 13 commercial antibodies targeting the neuronal synaptic vesicle protein Synaptotagmin-1. This is a protein essential for the fusion of synaptic vesicles and subsequent release of neurotransmitter into synaptic clefts, critical for neurotransmission in brain. Recent studies have identified novel loss of function mutations in patients suffering with neurodevelopmental delay underscoring the importance of developing tools for discovery purpose that accurately assess properties of Synaptotagmin-1 protein.\nAntibodies are critical as binder tools to identify and characterize protein localization and function in health and disease, but are often presented as “working” in contexts outside those of the intended user. As such much frustration is gained and money lost in trying to use these tools from commercial vendors.\nIn the current manuscript, Biddle et al, in conjunction with the YCharos collective present the results of a detailed evaluation of commercially available Synaptotagmin-1 antibodies using a carefully established pipeline. After identifying an immortal cell line that expresses high levels of Synaptotagmin-1 endogenously, and obtaining a Synaptotagmin-1 KO version of the same cell line, the authors proceed to assess antibody performance in a variety of applications including western blot, immunoprecipitation, cell immunofluorescent labelling and flow cytometry. The results clearly identify set(s) of antibodies that work for each assay and seem to correctly identify Synaptotagmin-1, as well as many that fail.\nThe manuscript is clearly and concisely written. The methodologies utilized seem appropriate. Importantly, a standardized set of methods are employed for westerns, immunoprecipitation and immunofluorescent labeling of cultured cells, a necessary part of establishing a platform with wide utility for the scientific community. These protocols are available online. The flow cytometry method is adequately detailed in the current manuscript. The results are clearly presented and support the contention that open source characterization of antibodies (commercial or otherwise) using a generalized set of methods with WT and KO conditions can be extremely valuable to the broader scientific community and provide a highly valuable resource for guiding future work. Work of this nature has high value.\nConcerns:\nFigure 3: The authors state they quantitatively measures immunofluorescence content in “hundreds” of cells immunolabelled with the various antibodies yet this data was not presented. As some of the antibodies do not appear to effectively label Synaptotagmin-1 its difficult to assess whether the labeling is affected in the KO cells. Quantitative numbers would help with assessment here.\nAlong same lines, the cell segmentation lines occlude assessment of the less effective labelers (eg mAb 30, 14558). Could they be limited to the DAPI panels? It’s unfortunate the WT and KO cells are not from the same source. Its unclear how this could impact subtle changes in protein expression based on passage number, culture conditions etc that could impact some Abs but not others. It would be more ideal to generate the KO and WT from same exact source of cells, although this may be beyond the capabilities of the collective. Alternatively, running a control Ab, not expected to be impacted by the KO could be used as a control for cell line “matching”.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "313882", "date": "20 Aug 2024", "name": "Jan Voskuil", "expertise": [ "Reviewer Expertise Life-time career expertise on antibody characterisation and validation in all immunoassays. Both experience as an academic scientist", "and as a commercial provider." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 delivered an excellent approach to validate commercial antibodies by comparing their generated signals from a suitable cell line, proven to express the target gene sufficiently for antibody detection, with its cognate knock-out derivative cell line.\nIn their approach, they analyse the antibody performance in Western blot (WB), in immuno-precipitation (IP), in immunocytochemistry (staining of entire cells detected by immunofluorescence, IF) and even in flow cytometry (FC).\nAlthough the objective of the authors are to present their approach as a method of picking a high performing antibody from the market, the data themselves are left without scientific assessment. I think this paper will benefit from an extra paragraph to discuss the quality of the individual data. For example, some antibodies show extra bands in WB, and it is not clear whether these bands would disappear upon further antibody dilution (without loss of specific signal), or that all the bands, the correct and the wrong ones, would equally fade by further antibody dilution. The target Synaptotagmin-1 is located in secretory vesicles and synaptic granules. Its location in the IF data is not very clear in many cases, located at the cellular membrane at best. A few sentences on the quality of the data would be desirable and put some extra scientific weight to the article. I would also like to read how the performance of the best antibodies compare across the different assays. It seems MAB4364 and 105011 are the best in FC, followed by 68043-1-Ig and 105008. But in IF the latter two outperform the former two. What is the authors' opinion on such inconsistency? Both assays are physically similar to one another and fundamentally different from WB and IP. All four antibodies perform extremely well in WB and IP.\n\nThe paper is extremely powerful and the first of its kind to rigorously assess the specificity and selectivity of commercial antibodies in such consistent way. I highly commend this paper for indexing, and I hope my remarks will be addressed in the final version.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-817
https://f1000research.com/articles/8-192/v1	15 Feb 19	{ "type": "Systematic Review", "title": "Optimizing the use of digital sensors (non-invasive) for early detection of risk factors for recurrent stroke to improve quality of care: A systematic review", "authors": [ "I Made Kariasa", "Elly Nurachmah", "Setyowati Setyowati", "Raldi Artono Koestoer", "Elly Nurachmah", "Setyowati Setyowati", "Raldi Artono Koestoer" ], "abstract": "Background: Improving awareness in patients with stroke to detect risk factors of recurrent stroke has recently become a major challenge for all health professionals in preventing recurrence among stroke survivors. Utilization of advanced technology, such as digital sensors, (a non-invasive device) is among the breakthroughs in detecting the risk of disease and promotes more effective prevention and improves quality of care. This study aims to conduct a systematic review on studies addressing early detection of the risk factors of recurrent stroke through the utilization of digital sensors. Methods: A literature search was carried out on articles published between 2011 - 2018 on EBSCO, Elsevier, Science Direct, ProQuest, Springer link, PubMed, MEDLINE, PLoS, and the Journal of Community Nursing. The search identified quantitative research studies on the utilization of digital sensors in cases of hypertension, diabetes mellitus, hypercholesterolemia, and cardiac disorders that contributed to stroke recurrence. Each study’s bias was assessed using Review Manager 5. Results:\nTen articles were analyzed through data extraction. Robust assessment of independent risk factors which provoke recurrent stroke such as hypertension, diabetes mellitus, hypercholesterolemia, and heart diseases could lead to conservation of health resources. It is very important to monitor these factors. With the development of technology, the potential use of non-invasive monitoring for hypertension, diabetes mellitus, hypercholesterolemia, and heart diseases as risk factors for recurrent stroke events is considered effective because it is easy, simple, low cost, sensitive, and does not cause additional suffering for patients. Conclusion: A practical and non-invasive method for early detection and monitoring of risk factors may reduce the risk of stroke recurrence among stroke survivors.", "keywords": [ "Digital sensor", "quality of care", "risk factors", "recurrent stroke." ], "content": "Introduction\n\nStroke is defined as an infarction of brain, spinal cord, or retinal cell death attributable to ischemia, based on pathological, imaging, or other objective evidence of cerebral, spinal cord, or retinal focal ischemic injury in a defined vascular distribution; or clinical evidence of cerebral, spinal cord, or retinal focal ischemic injury based on symptoms persisting ≥ 24 hours or until death, and other etiologies are excluded1. In 2013, strokes contributed to 1 out of 20 deaths in the United States. Stroke affects one person in every 40 seconds and it claims one life every 4 minutes2.\n\nThere are two types of risk factors for stroke: modifiable and non-modifiable. Non-modifiable risk factors include age, gender, low birth weight, race, and genetic factors. Modifiable risk factors include hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation, and heart disorders3. Patients who have suffered from strokes are at risk of stroke recurrence or relapse with a cumulative risk of 39.2%. Approximately 13% of patients with stroke are affected by recurrent stroke within the first year after the initial event4. A systematic review reported that recurrent stroke was the major reason for hospital re-admission among patients with ischemic stroke within the first 30 days and 1 year following the initial event, (33% and 26.3%, respectively)5. Hospitals are currently challenged to reduce preventable hospital re-admission for cost retrenchment and improvement of service quality. Furthermore, the incidence of recurrent stroke is significantly associated with increased mortality and morbidity because it results in greater neurological deficits than initial stroke, which lead to longer hospital stays and higher cost of care6.\n\nVarious prevention and behavior change programs have been implemented. However, these have not significantly reduced the prevalence of recurrent stroke and re-admission rate5. Health professionals are facing a challenge to provide health education to the community and make a breakthrough to improve awareness about stroke or recurrent stroke, especially awareness among those who possess higher risk factors. Utilization of advanced technology, such as digital sensors, (a non-invasive device) is among the breakthroughs in detecting the risk of disease for more effective prevention3. Unlike invasive procedures, sensor technology undoubtedly facilitates health professionals in detecting risk factors of stroke in patients in an easy and pain-free manner1.\n\nTo the best of the author’s knowledge, there has not been a study conducted that systematically reviews the literature addressing early detection of risk factors of stroke through the use of digital sensors in order to establish proper practice guidelines in developing non-invasive devices for identifying the risk factors for stroke recurrence.\n\n\nMethods\n\nThe search strategy included the following terms: 'risk factor of stroke', 'post-stroke', 'recurrent stroke', ‘stroke prevention’, 'detection', 'digital sensor', 'non-invasive', and 'nursing'. The chronological search range was between 1 January 2018 and 31 August 2018. Databases used for searching literature included: EBSCO, Elsevier ScienceDirect, ProQuest, Springer link, PubMed, MEDLINE, PLoS, and the Journal of Community Nursing.\n\nThe search process resulted in 92 articles which matched the keywords. These articles were then filtered according to full text and publication year between 2011 – 2018 which resulted in 49 articles. These 49 articles were then reviewed based on titles matching the use of digital sensors (non-invasive) for early detection of risk factors for recurrent stroke and resulted in 16 articles. Finally, these 16 articles were filtered based on inclusion and exclusion criteria, (see below) and resulted in 10 articles. The search process on the databases is shown in Figure 1.\n\nCritical appraisal was later performed according to the study design of the articles. The Critical Appraisal Skills Program (CSAP) was used for analysis7.\n\nTen articles were analyzed through data extraction. Variables acquired through data extraction were: authors name(s), year of study and publication, intervention method, and study findings. All the items were included in the table of data extraction. We used the Cochrane Collaboration free software called Review Manager (RevMan version 5.3)8 which is useful for analyzing results and generating forest plots and risk-of-bias. The results of bias assesed are shown in Figure 2.\n\n1. Stroke survivor.\n\n2. Finding: Patient identified as having a high risk factor for recurrent stroke.\n\n3. Include examination of stroke risk factors using digital sensors.\n\n4. Language and publication date: published in English language between 2011 and 2018.\n\n5. Study design: Randomized Controlled Trial, meta-analysis, cohort study, survey and case report.\n\nThis study that did not address the use of sensory non-invasive devices to detect risk factor of stroke and nursing intervention in educating patients to prevent stroke recurrence.\n\n\nResults\n\nThe characteristics of the selected studies are outlined in Table 1. The systematic review revealed risk factors of recurrent stroke that included hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation, and heart disorders. Those risk factors were detected by a non-invasive device consisting of various sensors and tools that reduced the patient’s pain during examination. The review also found that the nurse is part of the multidisciplinary team that provides care for patients with stroke. One of nurse’s roles and functions is to promote independence of the patient in performing self-screening, prevention, disease management and rehabilitation. Advancing technology would facilitate a nurse in identifying risk factors of stroke.\n\nUtilization of digital sensors to detect hypertension. Studies have suggested that hypertension is the main risk factor for stroke, though it is also the easiest to modify if the affected person is willing to adopt a healthier lifestyle. The mean blood pressure of the affected person ranges from systolic pressure ≥ 140 mmHg or diastolic pressure ≥ 90 mm Hg. Prevalence of hypertension in patients with ischemic stroke approaches 70%19. Hypertension treatment is described as the most crucial intervention in secondary prevention of ischemic stroke. The risk of ischemic stroke is significantly associated with blood pressure (BP) with systolic blood pressure (SBP) as low as 115 mmHg. It is also directly correlated with recurrent strokes. Therefore, regular monitoring will assist in providing appropriate medication17.\n\nPeople with hypertension require a blood pressure measurement device to monitor their hypertension status. However, they also need to access a health facility just to measure their blood pressure and the measurement is performed by a trained staff, such as a nurse or other health professional. It is considered a time consuming activity by patients who could otherwise be more productive. Although they may be able to purchase it, the device is mostly magnetically based and applies an indirect method of blood pressure detection using Korotkoff sounds. The method is also difficult to conceptualize and there are certain procedures that require careful attention, including proper cuff positioning which, significantly affects the measured parameter. Furthermore, the device also possesses built-in ambivalence in which the measurement of systolic and diastolic pressure from an empirically measured mean of pressure is also influenced by user’s hearing. Therefore, its use is rather less effective and efficient16. Such deficiencies have led to the development of a blood pressure measurement device that utilizes automatic measurement features. It is easy to use, only requiring a finger tap and the sensor will automatically measure the blood pressure with a sensitivity matching the user’s condition. Measurement of blood pressure through such a digital sensor also offers advantage that it enables the patient to monitor his own blood pressure at any time18.\n\nUtilization of digital sensor to detect blood glucose. Elevation of blood glucose levels over the normal range indicates diabetes mellitus which is an independent risk factor for stroke. Most people with diabetes are also affected by hypertension, hypercholesterolemia and are overweight. These conditions increase the risk for stroke recurrence. Even though diabetes is a treatable disease, uncontrolled diabetes may increase the risk for stroke17.\n\nControl of blood glucose levels heavily depends on blood glucose monitoring. Patients with type 1 or type 2 diabetes are recommended to check his or her blood glucose level up to 4 times a day14. The measurement is generally performed by using a finger-prick glucose meter. The test will reveal the patient’s blood glucose level which is required to calculate the insulin dose every day. The disadvantage of this measurement is that it causes pain when the finger is pricked. In addition to the impracticality of glucose meter device, there is also a risk for infection associated with tissue injury and damage of tissue associated with puncture wounds over a long period. Several factors may cause the patient to neglect blood glucose monitoring11. This reality led to the invention of non-invasive technologies for blood glucose monitoring. Advancing technology allows exploration of non-invasive blood glucose monitoring through the use of a practical digital sensor that is portable and pain-free for the user. Infrared spectroscopy is the method used by a type of sensor which is highly sensitive to blood glucose levels. The patient only needs to place a finger on the sensor and it will operate according to user’s physiological condition, metabolism, and circulation. This device requires reliability and must be calibrated to ensure the accuracy of the blood glucose result11.\n\nUtilization of digital sensor to detect cholesterol. People with hypercholesterolemia are at higher risk of stroke. A considerable amount of cholesterol in the blood might develop and lead to coagulation, atherosclerosis, and blood flow obstruction which causes stroke. Moreover, low levels of High Density Lipoprotein, (HDL “good” cholesterol) is recognized as a risk factor for stroke in males17. Modification of primary serum lipid biomarkers, such as low-density lipoprotein cholesterol (LDL-C), might be implemented as a strategy for reducing risk of secondary stroke among patients with TIA or ischemic stroke. Considering that hypercholesterolemia is associated with arteriosclerosis, hypertension, and myocardial infarction, identifying cholesterol levels is imperative for establishing a clinical diagnosis. A diagnostic test is required to identify cholesterol levels. Most hospitals currently still apply an invasive or traditional procedure that includes glass, ceramic and polymer and the results require waiting time. Therefore, a device to detect cholesterol levels through a digital sensor which is pain-free with rapid and accurate results is currently being developed15.\n\nDevelopment of a sensitive, accurate, and inexpensive biosensor will facilitate the filtering of biomarkers of certain diseases. Cellulose filter paper has recently been invented for sensory applications at a very low cost. The paper-based biosensor offers several advantages including biocompatibility, low cost and disposability10. In addition, amperometric biosensors that incorporate analytical devices are capable of converting concentrations of electrical signals which are integrated with biological sensors. Hence, portable biosensors are user-friendly, possess high specificity, and are currently utilized in multidisciplinary fields, including pharmacy, nursing, medicine and physiotherapy. The portable biosensors allow easy detection of cholesterol levels without any associated pain and require shorter times to produce the result. A sensitive, selective, reliable and inexpensive cholesterol sensing device is presently being developed for clinical use in measuring cholesterol levels which allows independent monitoring by the user at home12.\n\nUtilization of digital sensors to detect cardiac disorders. Heart attack occurs when plaque accumulation causes a blockage in blood vessels supplying the heart. Most stroke cases are also primarily caused by an accumulation of plaque that causes a blockage in blood vessels within the brain9. Electrocardiography (ECG) may be employed to identify heart disorders and its use is still very widespread. However, the result requires interpretation by health professionals, even if the patient is able to afford the device. A study reported that 1 out of 5 patients with ischemic stroke or TIA had a medical history of atrial fibrillation (AF) or indicated that they were affected by AF, according to a 12-lead ECG, putting at risk in the future. Early detection of AF may lead to appropriate treatment and offer more cost effective care in health system13.\n\n\nDiscussion\n\nRecurrent stroke is an event that occurs after an initial stroke attack. The American Stroke Association (ASA) claims that the average risk of stroke recurrence after ischemic stroke or TIA (transient ischemic attack) is 3–4%2. Following recuperation from stroke, most patients prioritize rehabilitation and recovery programs while neglecting efforts for secondary prevention. Secondary prevention is fundamental to prevent stroke recurrence as it may result in a more debilitating impact than the initial stroke3.\n\nRisk factors of recurrent stroke are similar to those of primary stroke and include modifiable and non-modifiable factors. Secondary prevention refers to all efforts made to prevent recurrent stroke. Secondary prevention involves all attempts which address modifiable risk factors20. Secondary prevention consists of risk factor management, intervention to resolve vascular obstruction, anti-thrombolytic therapy for cardio embolic stroke, and antiplatelet therapy for non-cardio embolic stroke. Nurses play an important role as educators in secondary prevention by providing health information for controlling risk factors5. Studies addressing knowledge about the risk of impending stroke had been focused on the community as a whole. A study evaluating knowledge about risk factor in patients at high risk revealed that only 42% of patients with a history of stroke were aware of their own risk of stroke recurrence and only 27% of them reported it to a physician. The study also identified a low level of awareness of risk of recurrence among high risk participants (41%), especially those with a medical history of hypertension, diabetes mellitus, hypercholesterolemia and heart disorders21.\n\nAssessment of independent risk factors which trigger recurrent stroke such as hypertension, diabetes mellitus, hypercholesterolemia and heart disease are resources intensive. The frequent measurement of blood glucose is an essential part of recurrent stroke monitoring22. Despite the fact that almost all the commercially successful blood glucose monitoring devices are invasive, there is an immense need to develop non-invasive monitoring devices that will alleviate the pain and suffering of patients associated with the frequent pricking of skin needed to obtain blood samples for glucose testing23. These issues may cause the patient to neglect blood glucose monitoring in health facilities. Therefore, all health professionals, including nurses, are required to be involved in monitoring the patient’s recovery process, including providing motivation and involvement in the development of health devices that contribute to an effective and efficient service for the patient. Current health devices are primarily developed based on a non-invasive concept, which aims to provide comfort during the diagnostic process and are more practical than invasive procedures3.\n\nA glucose sensor with ultrahigh sensitivity and a rapid response time was constructed using a PtNP/ PANI hydrogel heterostructure for non-invasive glucose monitoring14. Studies have shown that the glucose sensors composed of the PtNP/PANI hydrogel heterostructure exhibited unprecedented performance with ultrahigh sensitivity, fast response, and a very low detection threshold and has great potential for use in applications relating to medical diagnosis14. Another study about non-invasive glucose monitoring was conducted by Luo et al. (2011). In this study, a novel amperometric glucose sensor based on Cu decorated graphene sheets sensor the advantages of ease of fabrication, low cost, good reproducibility and perfect specificity to glucose, so is a potential candidate for routine glucose analysis11.\n\nSimilar to glucose, methods of monitoring non-invasive hypercholesterolemia are being developed. Ruecha et al. (2014) fabricated a novel nano composite based on G/PVP/PANI using a paper-based cholesterol biosensor that might be an alternative tool for cholesterol screening in medical diagnosis due to its simplicity, low cost, disposability and portability15. Other media such as field-effect transistors have a wide linear range and high sensitivity for detection of cholesterol which suggests that this cost-effective process can lead to portable, reliable and real-time cholesterol detection12. In addition, amperometic cholesterol biosensors using a layer-by-layer adsorption technique onto electrospun polyainiline nanofibers has been used to measure cholesterol consentration10.\n\nNon-invasive monitoring of heart disease as a reccurent stroke risk factor was conducted by Higgins et al. (2013) using the Novacor R-test Evolution 3 device to detect atrial fibrillation after ischemic stroke13. These method was effective because it enhances detection of paroxysmal atrial fibrilation and early anticoagulation13. Non-invasive blood pressure measurement using android smart phones and web applications offer solutions for patients, relatives, doctors and nurses to monitor blood pressure as a predictor of reccurent stroke16,18.\n\nHowever, most non-invasive technologies, especially digital sensors that detect blood pressure, blood glucose level, cholesterol level and cardiac disorders are still under development. There are various non-invasive technologies outlined in this study and the volume of new studies for this type of non-invasive concept increases yearly. The current situation requires constant updates of available technology. With the advancement of technology, it becomes challenging for the health workers to develop a strategy for monitoring risk factors reccurent stroke in a comprehensive manner21. To that end, this paper provides an overview of currently available non-invasive digital sensors for monitoring blood pressure, blood glucose level, cholesterol level and heart disorders. Furthermore, the adoption of non-invasive monitoring technology is increasing because it is easy to use, low cost, sensitive, and does not cause pain in the patients11,15.\n\nA practical test to detect risk factors of recurrent stroke is expected to be used as an initial guideline to promote a healthier lifestyle and behavior among patients. Individual resolve should be taken into consideration to increase the success rate of the stroke patient’s behavior in monitoring and managing risk factors as well as modifying their lifestyle in order to prevent stroke recurrence which, in turn, improves his or her quality of life24. A previous article revealed that non-invasive methods of diagnostic tests should always be accompanied by an invasive method, specifically when identifying lesions in acute coronary syndrome and also in major diagnostic tests in order to prevent gaps in results from both methods24. However, several articles reported that application of non-invasive devices such as digital sensors offer ease of use, low cost, sensitivity, and do not cause suffering during detection of risk factors of stroke recurrence, including hypertension, diabetes mellitus, hypercholesterolemia and cardiac disorders11,15. Hence, development of non-invasive devices with valid predictive results progresses in order to drastically reduce invasive procedures that may cause pain for patients, especially in diagnostic tests for heart failure, kidney failure and vascular disorders25,26. Nevertheless, non-invasive devices have their weakness, and there are no digital sensors currently in use for the detection of hypertension, diabetes mellitus, hypercholesterolemia and heart disorders together.\n\nIt becomes a challenge for health workers to develop a strategy for monitoring risk factors for reccurent stroke in a comprehensive manner. On the other hand, the patient is required to purchase all four devices to identify risk factors of recurrent stroke. Yet, this self-screening ability will provide confirmation for patients who are at risk of stroke recurrence so they can follow it up with appropriate prevention measures, including improving lifestyle and accessing health provision to manage and mitigate the risk of recurrence. Recognizing changes in and development of risk factors in patients may inform their decision to access hospital treatment. Consequently, a non-invasive device capable of detecting the four risk factors of stroke recurrence in a single test is expected to be developed in the future. Moreover, such an all-in-one device would be able to calculate cumulative risk of recurrent stroke.\n\n\nConclusion\n\nThe incidence of recurrent stroke is quite high and causes re-hospitalization of patients. There are several factors that can cause recurrent stroke, including hypertension, hyperglycemia, hypercholesterolemia and heart disease especially atrial fibrillation. It is very important to monitor these factors. With the development of technology, the potential use of non-invasive monitoring for risk factors for recurrent stroke events is considered effective because it is easy to use, low cost, sensitive, and does not cause suffering in patients. There is currently no non-invasive monitoring that assesses all these factors simultaneously. The challenge for health workers is to develop a means of non-invasive monitoring of risk factors for the incidence of recurrent stroke in a comprehensive manner.\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: PRIMSA checklist, https://doi.org/10.17605/OSF.IO/DA3CK27.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis systematic review was funded by University of Indonesia with contract number of 1255/UN2.R3.1/HKP.05.00/2018.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nVishinkin R, Haick H: Nanoscale Sensor Technologies for Disease Detection via Volatolomics. Small. 2015; 11(46): 6142–6164. Accessed June 11th 2018. PubMed Abstract \| Publisher Full Text\n\nAdams RJ, Albers G, Alberts MJ, et al.: Update to the AHA/ASA recommendations for the prevention of stroke in patients with stroke and transient ischemic attack. Stroke. 2008; 39(5): 1647–1652. Accessed February 10th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence M, Kerr S, Watson HE, et al.: A survey of stroke nurses’ knowledge of secondary prevention lifestyle issues. Br J Neurosci Nurs. 2009; 5(11): 518–523. Accessed February 10th 2018. Publisher Full Text\n\nEvans-Hudnall GL, Stanley MA, Clark AN, et al.: Improving secondary stroke self-care among underserved ethnic minority individuals: a randomized clinical trial of a pilot intervention. J Behav Med. 2014; 37(2): 196–204. Accessed May 11th 2018. PubMed Abstract \| Publisher Full Text\n\nRao A, Barrow E, Vuik S, et al.: Systematic Review of Hospital Readmissions in Stroke Patients. Stroke Res Treat. 2016; 2016: 9325368. Accessed May 15th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVest JR, Gamm LD, Oxford BA, et al.: Determinants of preventable readmissions in the United States: a systematic review. Implement Sci. 2010; 5(1): 88. Accessed May 20th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCritical Appraisal Skills Programme (CASP): Critical Appraisal Skills Programme (CASP). [online]. 2015; Accessed June 10th 2018. Reference Source\n\nRevMan 5.3 User Guide. [online]. Accessed Des 10th 2018. Reference Source\n\nKirschbaum SW, Nieman K, Springeling T, et al.: Non-invasive diagnostic workup of patients with suspected stable angina by combined computed tomography coronary angiography and magnetic resonance perfusion imaging. Circ J. 2011; 75(7): 1678–84. Accessed January 21th 2018. PubMed Abstract \| Publisher Full Text\n\nShin YJ, Kameoka J: Amperometric cholesterol biosensor using layer-by-layer adsorption technique onto electrospun polyaniline nanofibers. J Ind Eng Chem. 2012; 18(1): 193–197. Accessed May 10th 2018. Publisher Full Text\n\nLuo J, Jiang S, Zhang H, et al.: A novel non-enzymatic glucose sensor based on Cu nanoparticle modified graphene sheets electrode. Anal Chim Acta. 2012; 709: 47–53. Accessed February 10th 2018. PubMed Abstract \| Publisher Full Text\n\nAhmad R, Tripathy N, Hahn YB: High-performance cholesterol sensor based on the solution-gated field effect transistor fabricated with ZnO nanorods. Biosens Bioelectron. 2013; 45: 281–286. Accessed May 11th 2018. PubMed Abstract \| Publisher Full Text\n\nHiggins P, MacFarlane PW, Dawson J, et al.: Noninvasive cardiac event monitoring to detect atrial fibrillation after ischemic stroke: a randomized, controlled trial. Stroke. 2013; 44(9): 2525–2531. Accessed February 11th 2018. PubMed Abstract \| Publisher Full Text\n\nZhai D, Liu B, Shi Y, et al.: Highly sensitive glucose sensor based on pt nanoparticle/polyaniline hydrogel heterostructures. ACS Nano. 2013; 7(4): 3540–3546. Accessed May 11th 2018. PubMed Abstract \| Publisher Full Text\n\nRuecha N, Rangkupan R, Rodthongkum N: Novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite. Biosens Bioelectron. 2014; 52: 13–19. Accessed February 10th 2018. PubMed Abstract \| Publisher Full Text\n\nIlango S, Sridhar P: A Non-Invasive Blood Pressure Measurement using Android Smart Phones. IOSR J Dent Med Sci. 2014; 13(1): 28–31. Accessed May 14th 2018. Publisher Full Text\n\nXuan W, Pan R, Wei Y, et al.: Reaction-Based ‘Off−On’ Fluorescent Probe Enabling Detection of Endogenous Labile Fe2+ and Imaging of Zn2+-induced Fe2+ Flux in Living Cells and Elevated Fe2+ in Ischemic Stroke. Bioconjugate Chem. 2016; 27(2): 302–308. Accessed February 11th 2018. Publisher Full Text\n\nVillarreal V, Nielsen M, Samudio M: Sensing and Storing the Blood Pressure Measure by Patients through A Platform and Mobile Devices †. Sensors (Basel). 2018; 18(6): pii: E1805. Accessed July 10th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFurie KL, Kasner SE, Adams RJ, et al.: Guidelines for the prevention of stroke in patients with stroke or transient ischemic attack: a guideline for healthcare professionals from the american heart association/american stroke association. Stroke. 2011; 42(1): 227–76. Accessed February 4th 2018. PubMed Abstract \| Publisher Full Text\n\nLeoo T, Lindgren A, Petersson J, et al.: Risk factors and treatment at recurrent stroke onset: results from the Recurrent Stroke Quality and Epidemiology (RESQUE) Study. Cerebrovasc Dis. 2008; 25(3): 254–260. Accessed July 11th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSo CF, Choi KS, Wong TK, et al.: Recent advances in noninvasive glucose monitoring. Med Devices (Auckl). 2012; 5: 45–52. Accessed February 4th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlark J, Sharma P: Risk awareness in secondary stroke prevention: a review of the literature. JRSM Cardiovasc Dis. 2014; 3: 204800401351473. Accessed February 4th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVashist SK: Non-invasive glucose monitoring technology in diabetes management: a review. Anal Chim Acta. 2012; 750: 16–27. Accessed July 11th 2018. PubMed Abstract \| Publisher Full Text\n\nLawrence M, Pringle J, Kerr S, et al.: Multimodal secondary prevention behavioral interventions for TIA and stroke: a systematic review and meta-analysis. PLoS One. 2015; 10(3): e0120902. Accessed February 4th 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagai K, Shibata S, Akishita M, et al.: Efficacy of combined use of three non-invasive atherosclerosis tests to predict vascular events in the elderly; carotid intima-media thickness, flow-mediated dilation of brachial artery and pulse wave velocity. Atherosclerosis. 2013; 231(2): 365–370. Accessed July 11th 2018. PubMed Abstract \| Publisher Full Text\n\nSacco RL, Kasner SE, Broderick JP, et al.: An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2013; 44(7): 2064–2089. Accessed February 4th 2018. PubMed Abstract \| Publisher Full Text\n\nKariasa IM; F1000 Research: PRISMA CECKLIST. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/DA3CK" }	[ { "id": "69142", "date": "18 Aug 2020", "name": "Loo Keat Wei", "expertise": [ "Reviewer Expertise Meta-analysis", "ischemics stroke", "bioinformatics", "computational epigenetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHow to define “early detection of the risk factors” when conceptualizing the paper.\n\nThe “proper practice guidelines” were listed as part of the objective, however, it hasn't been discussed and described thoroughly in the manuscript.\n\nThe year of publication was limited to 2011-2018. There are couples of new studies 2019-2020, why such studies were not included?\n\nThe literatures searching were performed through a combination of indexing databases and journal databases, with majority of the searching were done through journal databases. Why was such a searching done? It is advisable to the author to search through the indexing databases rather than the journal databases. By which, the results obtained through the searching on these limited numbers of journal databases is incomplete.\n\nThe search term is quite diversified, ‘risk factor of stroke’, ‘post-stroke’, ‘recurrent stroke’, ‘stroke prevention’, ‘detection’, ‘digital sensor’, ‘non-invasive’, and ‘nursing’.” Not sure why nursing was used as part of the search term. Whilst the title involves quality of care, and such term was not included as part of the search term?\n\nThe article would be much more comprehensive if the author can include the risk factors such as “hypertension”, “diabetes”, etc. as part of the search term.\n\nFigure 1 shows the process of screening. It is advisable that the author should include the items that have been excluded in Figure 1. It has no meaning to input the number as n=43, without telling the reader why it is excluded.\n\nI found these sentences very confusing “The search process resulted in 92 articles which matched the keywords. These articles were then filtered according to full text and publication year between 2011 – 2018 which resulted in 49 articles. These 49 articles were then reviewed based on titles matching the use of digital sensors (non-invasive) for early detection of risk factors for recurrent stroke and resulted in 16 articles. Finally, these 16 articles were filtered based on inclusion and exclusion criteria, (see below) and resulted in 10 articles. The search process on the databases is shown in Figure 1.” If there are 92 articles that matched the keywords, shouldn’t all the 92 articles being screen through the inclusion/exclusion criteria? Why the author needs to review the title that matched with digital sensors, then screen the title based in the inclusion/exclusion criteria?\n\nThe inclusion criteria of study design: Randomized Controlled Trial, meta-analysis, cohort study, survey and case report. However, “experimental” has been used in Table 1. Thus, it is not sure whether experimental work is referring to survey/case report/ any other types of study design.\n\nTo increase the transparency of the quality assessments, the information of Critical Appraisal Skills Program (CSAP) should be appended as supplementary files.\n\nPlease elaborate more on “Ten articles were analyzed through data extraction.”\n\nThe meta-analysis has been used as the inclusion criteria for the study. How many meta-analysis papers have been included in the analysis so far? Shouldn’t the meta-analysis paper being excluded from the analysis?\n\nNursing intervention has been used as the exclusion criteria for the study, but the search term “nursing” has been used in the literature searching. It is contradicting. Are digital sensors the same as digital devices? Are they interchangeable? Any differences between them? As illustrated by the author, blood pressure measurement device has been used to monitor their hypertension status. Such deficiencies have led to the development of a blood pressure measurement device that utilizes automatic measurement features. …..It is easy to use, only requiring a finger tap and the sensor will automatically measure the blood pressure with a sensitivity matching the user’s condition. Measurement of blood pressure through such a digital sensor also offers advantage that it enables the patient to monitor his own blood pressure at any time18. The author is describing only the digital sensors/digital device for BP monitoring?\n\nPlease elaborate more on “Therefore, a device to detect cholesterol levels through a digital sensor which is pain-free with rapid and accurate results is currently being developed15.”\n\nAs for the biosensors, have they been verified clinically? Or they are still in the stages of clinical trials?\n\nIt would be of great interest for the clinicians and researchers, if the author could include the biosensors which have been verified clinically. Otherwise, the findings of this systematic review need to be analyzed with caution.\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": "11837", "date": "19 Jul 2024", "name": "I Made Kariasa", "role": "Author Response", "response": "1. How to define “early detection of the risk factors” when conceptualizing the paper. We have added the explanation of early detection of the risk factors. 2. The “proper practice guidelines” were listed as part of the objective, however, it hasn't been discussed and described thoroughly in the manuscript. We have revised the objective. 3. The year of publication was limited to 2011-2018. There are couples of new studies 2019-2020, why such studies were not included? “The chronological search range was between 1 January 2018 and 31 August 2018.” 4.The literatures searching were performed through a combination of indexing databases and journal databases, with majority of the searching were done through journal databases. Why was such a searching done? It is advisable to the author to search through the indexing databases rather than the journal databases. By which, the results obtained through the searching on these limited numbers of journal databases is incomplete. We used that based on the full-text database that we can access. 5. The search term is quite diversified, ‘risk factor of stroke’, ‘post-stroke’, ‘recurrent stroke’, ‘stroke prevention’, ‘detection’, ‘digital sensor’, ‘non-invasive’, and ‘nursing’.” Not sure why nursing was used as part of the search term. Whilst the title involves quality of care, and such term was not included as part of the search term? After a comprehensive discussion, we removed term ‘nursing’ and ‘quality of care’. 6. The article would be much more comprehensive if the author can include the risk factors such as “hypertension”, “diabetes”, etc. as part of the search term. Thank you for your constructive suggestions. The author used term “stroke risk factors” and identified various factors that caused recurrent attacks. From this finding the author was able to conclude that those causing recurrent attacks were sufferers who had a history of hypertension, diabetes, cholesterol and heart problems. So this finding is highlighted by the current author. 7. Figure 1 shows the process of screening. It is advisable that the author should include the items that have been excluded in Figure 1. It has no meaning to input the number as n=43, without telling the reader why it is excluded. We have added the explanation 8. I found these sentences very confusing “The search process resulted in 92 articles which matched the keywords. These articles were then filtered according to full text and publication year between 2011 – 2018 which resulted in 49 articles. These 49 articles were then reviewed based on titles matching the use of digital sensors (non-invasive) for early detection of risk factors for recurrent stroke and resulted in 16 articles. Finally, these 16 articles were filtered based on inclusion and exclusion criteria, (see below) and resulted in 10 articles. The search process on the databases is shown in Figure 1.” If there are 92 articles that matched the keywords, shouldn’t all the 92 articles being screen through the inclusion/exclusion criteria? Why the author needs to review the title that matched with digital sensors, then screen the title based in the inclusion/exclusion criteria? We have revised the paragraphs. 9. The inclusion criteria of study design: Randomized Controlled Trial, meta-analysis, cohort study, survey and case report. However, “experimental” has been used in Table 1. Thus, it is not sure whether experimental work is referring to survey/case report/ any other types of study design. We have revised it. Experimental refer to rct and non-rct 10. To increase the transparency of the quality assessments, the information of Critical Appraisal Skills Program (CSAP) should be appended as supplementary files. Thank you for your suggestion. We have added it. 11. Please elaborate more on “Ten articles were analyzed through data extraction.” Thank you for your suggestions. We have revised it. 12. The meta-analysis has been used as the inclusion criteria for the study. How many meta-analysis papers have been included in the analysis so far? Shouldn’t the meta-analysis paper being excluded from the analysis? Thank you for your confirmation. After a comprehensive discussion, we agreed to remove the meta-analysis because there is no meta-analysis study was used in the study. 13. Nursing intervention has been used as the exclusion criteria for the study, but the search term “nursing” has been used in the literature searching. It is contradicting. We have removed it. 14. Are digital sensors the same as digital devices? Are they interchangeable? Any differences between them? As illustrated by the author, blood pressure measurement device has been used to monitor their hypertension status. Such deficiencies have led to the development of a blood pressure measurement device that utilizes automatic measurement features. …..It is easy to use, only requiring a finger tap and the sensor will automatically measure the blood pressure with a sensitivity matching the user’s condition. Measurement of blood pressure through such a digital sensor also offers advantage that it enables the patient to monitor his own blood pressure at any time18. The author is describing only the digital sensors/digital device for BP monitoring? Digital sensors, often known as devices, capture physical or chemical phenomena/quantities present in the control system. A sensor operates by detecting occurrences and transmitting data that may be analyzed or identified by the controller. Electrical quantities are used for the purpose of detecting or measuring. When considering human users, they can rely on their five senses as sensors. In this context, the author explores how the skin acts as a mediator for these senses, allowing individuals to assess the condition of their body through sensory input. In addition, the author has included an explanation on the utilization of sensors for monitoring blood sugar levels, cholesterol levels, and cardiac conditions. The functioning principle of the sensor remains mostly consistent, with variations in the technology employed contingent upon the specific physiological system under consideration. 15. Please elaborate more on “Therefore, a device to detect cholesterol levels through a digital sensor which is pain-free with rapid and accurate results is currently being developed15.” Thank you for your suggestions. We have revised it. 16. As for the biosensors, have they been verified clinically? Or they are still in the stages of clinical trials? According to the author's examination of the literature, biosensors are shown to be precise in measuring cholesterol levels in human blood when compared to invasive methods. Similarly, biosensors have excellent sensitivity in assessing human blood sugar levels. 17. It would be of great interest for the clinicians and researchers, if the author could include the biosensors which have been verified clinically. Otherwise, the findings of this systematic review need to be analyzed with caution. Noted it well. It would be our further investigation." } ] }, { "id": "73527", "date": "18 Nov 2020", "name": "José Luis Ruiz-Sandoval", "expertise": [ "Reviewer Expertise Neurologist", "Stroke", "Intracerebral hemorrhage" ], "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\nDue to:\nIn Methods: Search strategy is inadequate: A cross analysis with “secondary stroke prevention” or “stroke recurrence” is lacking.\n\nThat references 10 to 18 correspond to studies about (mostly digital sensors) and viability, effectiveness or plausibility in detecting the most prevalent cardiovascular risk factors. None of the references are intended to specify the effect of these devices on secondary stroke prevention.\nI suggest: Change the title and objective (s) of the review. For example: “Feasibility and effectiveness of digital sensors (non-invasive) in monitoring the most prevalent cardiovascular risk factors”\nMoreover:\nReference 1 does not correspond to the content of the information described in the Introduction section.\nReference 17 does not correspond to the content of the information described in the Results section.\nComputed Tomography Coronary Angiography (CTCA) and Magnetic Resonance perfusion are not digital sensors devices.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "11838", "date": "19 Jul 2024", "name": "I Made Kariasa", "role": "Author Response", "response": "1. In Methods: Search strategy is inadequate: A cross-analysis with “secondary stroke prevention” or “stroke recurrence” is lacking. Thank you for your critical suggestion. We have revised our method 2. That references 10 to 18 correspond to studies about (mostly digital sensors) and viability, effectiveness or plausibility in detecting the most prevalent cardiovascular risk factors. None of the references are intended to specify the effect of these devices on secondary stroke prevention. Thank you for your critical opinion. I would like to clarify that hypertension, blood glucose, and cholesterol as the primary and secondary prevention. The study wants to investigate digital sensors and non-invasive devices that measure those three high-risk factors of recurrent stroke. 3. I suggest: Change the title and objective (s) of the review. For example: “Feasibility and effectiveness of digital sensors (non-invasive) in monitoring the most prevalent cardiovascular risk factors” Thank you for your suggestion. We modified the title. 4. Reference 1 does not correspond to the content of the information described in the Introduction section. Thank you for your suggestion. We have revised it. 5. Reference 17 does not correspond to the content of the information described in the Results section. Thank you for your suggestion. We have revised it. 6. Computed Tomography Coronary Angiography (CTCA) and Magnetic Resonance perfusion are not digital sensor devices. CTCA is a non-invasive detector-based spectral scanner. Our inclusion was digital sensors or non-invasive devices" } ] } ]	1	https://f1000research.com/articles/8-192
https://f1000research.com/articles/12-1340/v1	16 Oct 23	{ "type": "Research Article", "title": "Bibliometric analysis of scientific production on university social responsibility in Latin America and the Caribbean", "authors": [ "Diego Urrunaga-Pastor", "Guido Bendezu-Quispe", "Deici Dávila-Altamirano", "Milagritos N. Asmat", "Jordi Grau-Monge", "Guido Bendezu-Quispe", "Deici Dávila-Altamirano", "Milagritos N. Asmat", "Jordi Grau-Monge" ], "abstract": "Objective: To evaluate the scientific production on university social responsibility (USR) from institutions in Latin America and the Caribbean. Methods: A bibliometric analysis was conducted on documents published in indexed journals in the Scopus database from its inception until April 2023. Eligible documents included those on USR describing experiences carried out by universities in Latin America and the Caribbean. The number of articles per author, average authors per article, average citations per article, and the number of documents with one or more author were described. Bibliometric indicators regarding authors per article, co-authors per article, and institutional collaboration were presented. Bibliometric networks were constructed based on bibliographic coupling analysis of documents by countries and term co-occurrence in titles and abstracts. Results: Of a total of 4075 documents retrieved from Scopus, 150 were included. Documents published between 1997 and 2023 were identified, with an average annual growth rate of 2.7%. A total of 439 authors were identified, 18 articles had a single author, and an average of 0.3 articles per author and a co-authorship index of 3.13 were found. The percentage of international collaborations was 30.7%. Brazil had the highest proportion of publications (26.4%), followed by Chile (17%) and Colombia (13.2%). Opción and Revista de Ciencias Sociales were the journals with the highest number of articles published (13 each). In the analysis of term co-occurrence, recent years showed an increase in the use of terms related to e-learning, information and communication technologies, virtual education, COVID-19, sustainable development goals, and URSULA (initiative on USR in institutions in Latin America and the Caribbean). Conclusions: A growth in scientific production on USR in Latin America and the Caribbean was identified. The interest in USR documents in recent years has been focused on COVID-19 and the challenges of virtual education and sustainable development.", "keywords": [ "University social responsibility", "Latin America", "University", "Higher education", "Community" ], "content": "Introduction\n\nIn recent decades university social responsibility (USR) has gained increasing importance both in the academic and social spheres.1 USR refers to the commitment of higher education institutions to actively contribute to the sustainable development of society through the generation and application of knowledge, as well as the training of ethical and responsible professionals.1–4 It is essential to study USR to understand the impact of universities on their environment and foster greater integration between academia and society.5 Research on USR provides valuable insights into practices, challenges, and opportunities related to social responsibility in the university context.6\n\nThe scientific literature has addressed USR from different perspectives, but it is necessary to specifically analyze the scientific production in Latin American universities. Several previous studies have analyzed the topic of USR in different regions of the world, such as Europe and globally.7–12 However, research focused on Latin America and the Caribbean still presents significant gaps in terms of scientific production in this field. Understanding the current situation and trends in USR in Latin American universities will help identify areas for improvement and best practices, as well as provide a solid foundation for decision-making and policy formulation.7,13 Therefore, the aim of this study was to evaluate the scientific production in USR related to institutions in Latin America and the Caribbean, through bibliometric analysis of publications indexed in the Scopus database. It is expected to identify possible gaps and challenges in current scientific production, which will contribute to guiding future research and promoting academic collaboration in the region.5,14\n\n\nMethods\n\nA bibliometric analysis was conducted on the scientific production of USR in institutions from Latin America and the Caribbean. Scientific articles indexed in the Scopus database were included from its inception until April 2023. Articles addressing USR and describing an experience conducted by a university in Latin America and the Caribbean were included, regardless of the study design.\n\nThe Scopus database was chosen for the present bibliometric analysis due to its breadth and coverage of scientific articles, as well as its availability of metadata useful for this type of analysis.\n\nThe search strategy was conducted by one of the authors (GBQ) and included free terms used for searching the title, abstract, and keywords of the articles indexed in the Scopus database. The search formula is described in detail in a repository.15 The search strategy was independently reviewed and evaluated by another author (DUP). The search was not restricted by year or language of publication.\n\nData for each research article found during the search were downloaded as a.csv file from Scopus and imported into the Rayyan website. Two authors (DUP and GBQ) reviewed the title and abstract of each article to assess compliance with the eligibility criteria.\n\nThe Bibliometrix package in the R Studio statistical program was used for the analysis. One author (GBQ) manually standardized the names and affiliations of the authors found. The characteristics of the included articles were described, including the number of articles per author, average number of authors per article, average number of citations per article, and the number of documents with one or more author. Additionally, useful indicators for bibliometric evaluation, such as the authorship rate (ratio between the total number of articles and the total number of authors), co-authorship rate (average number of co-authors per article), and collaboration rate (ratio between the total number of authors of articles with multiple authors and the total number of articles with multiple authors) were described.\n\nThe VOSviewer software was used to construct and observe bibliometric networks based on bibliographic coupling analysis, using information on the countries of institutions with documents on USR and the co-occurrence of terms in titles and abstracts. No threshold was set for the inclusion of terms for the analysis of term co-occurrence. For this analysis, terms related to USR were excluded.\n\nData from documents published in journals indexed in the Scopus bibliographic database, which do not include confidential data of human subjects, were analyzed. Therefore, ethical committee approval was not required for this study.\n\n\nResults\n\nA total of 4,075 records were retrieved from the SCOPUS bibliographic database. After excluding documents that did not meet the eligibility criteria, 150 scientific articles were included (Figure 1). We included articles published between 1997 and 2023 (Figure 2). An increase in the number of scientific articles on USR was identified, with an average annual growth rate of 2.7%. The year 2022 had the highest number of published documents.\n\nRegarding the authors of the documents, a total of 439 authors with 469 mentions were identified. On average, there were 0.3 articles per author, a co-authorship index per document of 3.13, and 18 articles had a single author. The percentage of international collaborations was 30.7%. The authors Severino-González P (12 articles) and Sarmiento-Peralta G (4 articles) had the highest number of scientific publications (Table 1).\n\nIn terms of the country of origin of the institutional affiliation of the authors of the articles on USR, Brazil had the highest proportion of publications (26.4%), followed by Chile (17%) and Colombia (13.2%). Brazil had the highest number of documents as the sole country of institutional affiliations of the authors. Additionally, Brazil topped the list of countries with the highest number of citations and average citations per article, with 58 and 4.1, respectively, followed by Chile (49 and 5.4, respectively) and Colombia (45 and 6.4, respectively) (Table 2). These three countries (Brazil, Chile, and Colombia), along with Peru, were of note in international network collaboration in publications on USR (Figure 3).\n\nRegarding citations, the articles had an average of 4.6 citations. The articles by Sánchez-Hernández MI (2016) and Vallaeys F (2019) had the highest number of citations (42 and 36, with an average citation rate of 5.3 and 7.2 per year, respectively). Opción and Revista de Ciencias Sociales were the journals in which the highest number of articles were published, with 13 each (Table 3).\n\nIn the co-occurrence analysis of terms (Figure 4), it was identified that in the early years, publications on USR referred to terms such as ethics, knowledge, and teaching. In more recent years, the use of terms related to e-learning, information and communication technologies, virtual education, COVID-19, sustainable development goals, and URSULA (initiative on USR in Latin American and Caribbean institutions) became evident.\n\n\nDiscussion\n\nIn this study, we evaluated and characterized the scientific production related to USR by institutions in Latin America and the Caribbean using the Scopus database. We found an increase in scientific production over the past ten years, reaching a peak in 2022. According to the co-occurrence analysis of terms, recent documents on USR encompass topics related to the use of information and communication technologies, COVID-19, and sustainable development.\n\nThe observed increase in scientific production on USR by institutions in Latin America and the Caribbean could be attributed to a growing social awareness and commitment, manifested through increased investment and recognition by higher education institutions in social development. Additionally, due to significant social inequalities and challenges in areas such as poverty, education, health, and the environment, institutions in the region may show a greater interest in USR.16–18 Promoting research in this region could be facilitated by establishing close and collaborative partnerships between communities and universities. This entails working together to identify and address social issues and promote sustainable development.6 Furthermore, we identified peaks in scientific production between 2019 and 2022, which could be attributed to an upsurge in scientific output related to the COVID-19 pandemic.19–21 This is evident in the co-occurrence analysis of terms, where the term \"COVID-19\" is of note as one of the most frequently mentioned.\n\nThere was a predominance of countries such as Brazil, Chile, and Colombia in the publication of documents on USR by institutions in Latin America and the Caribbean. This finding aligns with the scientific production of these countries, which lead in research output in the region.22–24 It is worth noting that both the countries with the highest and lowest contributions to scientific production are of middle and high income, indicating no significant difference between income level and scientific contribution on USR. These results contrast with findings from other studies that have examined this potential relationship between income and scientific production.25 Therefore, it is possible that country-level promotion and initiatives explain this finding in countries with higher scientific production.\n\nThe authors with the highest scientific production did not exceed 13 scientific articles in Scopus, with researchers affiliated with institutions in Chile, Peru, and Colombia being of note, respectively. These countries, along with Brazil, demonstrate clear regional collaboration as well as collaboration with countries with advanced scientific development, as shown in the collaborative network graph. In various fields of knowledge, north-south collaboration is described as part of the early research development process in countries with lower scientific development.26,27 This influence may be observed in the development of research on USR in the Latin American and Caribbean region. On the other hand, the gap between the top-producing author and the second highest is eight articles, which reinforces the idea that USR is a young and growing field.\n\nThe scientific journal with the highest number of articles published was Opción, followed by the Revista de Ciencias Sociales and the Revista Venezolana de Gerencia. These journals publish papers related to social sciences, humanities, and education. This is consistent with a previous study,28 which explains that USR primarily falls within the field of social sciences, education, and humanities. Therefore, documents on USR from institutions in Latin America and the Caribbean would be of interest to these journals and their readers. All three journals are Venezuelan and have been indexed in Scopus for no more than 15 years. This aligns with the years when an increase in scientific production related to USR in Latin American institutions was observed.\n\nThe co-occurrence analysis of terms indicates that documents on USR from Latin American and Caribbean institutions have shifted from general aspects, such as ethics and education, to focusing on current challenges, such as the context of the COVID-19 pandemic, virtual education, and the use of information and communication technologies. As described in other fields, it is expected that over the years publications on a topic, such as USR, would shift focus from generalities to addressing its current applicability and challenges. Among the recently used terms is URSULA (Latin American Union for University Social Responsibility), which seeks to provide innovative proposals to improve the social and environmental role of universities through dialogue among different stakeholders, such as civil society, governments, scientists, and businesses.29 In recent years, some Latin American countries have worked on implementing policies and programs that promote USR.6,14,30 These initiatives may include incentives, funding, or specific requirements for academic institutions to conduct research and projects aimed at social well-being. This situation, combined with increased access to resources and technology in academic institutions, would generate greater opportunities for scientific research and the dissemination of its results, which could also explain the increase in scientific production on USR in the region.\n\nTo the best of our knowledge, this bibliometric analysis is the first to evaluate the scientific production on USR associated with institutions in Latin America. The results are valuable for identifying the current status and growth of this area. Additionally, we can identify authors who work in this field and the journals that frequently publish related articles. Among the potential limitations of this research, it should be mentioned that articles published in databases other than Scopus were not included, which means that some documents on USR from institutions in Latin America and the Caribbean may not have been considered. However, we believe that using Scopus, a reliable and widely employed source for bibliometric studies, ensures that the results obtained are from documents published in journals with quality criteria, such as peer review, and the requirements demanded by the bibliographic database for the inclusion of indexed journals, that is, the documents retrieved through the search strategy in this study. In this bibliometric analysis, a review of the title and abstract of the documents was conducted for their inclusion in the analysis, which strengthens the study as it ensured the inclusion of only documents on USR.\n\n\nConclusions\n\nIn conclusion, there is an increase in scientific production on USR by researchers from institutions in Latin America and the Caribbean. The countries leading the research in the region also show leadership in documents on USR. In recent years, the focus of documents on USR has been on COVID-19, virtual education, and sustainable development. Quantifying the scientific production on USR in Latin America and the Caribbean provides a baseline for future research in the field.", "appendix": "Data availability\n\nFigshare: Search strategy. DOI: https://doi.org/10.6084/m9.figshare.24179106. 15\n\nThis project contains the following underlying data:\n\n• An.docx file containing Scopus search strategy used to perform this bibliometric analysis.\n\nFigshare: Database containing the articles included in the analysis. DOI: https://doi.org/10.6084/m9.figshare.24069399. 31\n\nThis project contains the following underlying data:\n\n• An.xls file containing the database of articles included in the bibliometric analysis is available.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\n\nAcknowledgment\n\nWe would like to express our gratitude to the Universidad Científica del Sur for their English editing support and financial assistance in covering the article processing charge.\n\n\nReferences\n\nAli M, Mustapha I, Osman S, et al.: University social responsibility: A review of conceptual evolution and its thematic analysis. J. Clean Prod. 2021; 286: 124931. Publisher Full Text\n\nCoelho M, Menezes I: University social responsibility as a driving force of change: students’ perceptions beyond the ivory tower. On the Horizon. 2020; 28(2): 93–100. Publisher Full Text\n\nKouatli I: The contemporary definition of university social responsibility with quantifiable sustainability. Social responsibility journal. 2019; 15(7): 888–909. Publisher Full Text\n\nSantos G, Marques CS, Justino E, et al.: Understanding social responsibility’s influence on service quality and student satisfaction in higher education. J. Clean Prod. 2020; 256: 120597. Publisher Full Text\n\nVasilescu R, Barna C, Epure M, et al.: Developing university social responsibility: A model for the challenges of the new civil society. Procedia-Social and Behavioral Sciences. 2010; 2(2): 4177–4182. Publisher Full Text\n\nMartí-Noguera JJ, Martí-Vilar M: Social responsibility in basic and higher education: An approach for Latin-America. Revista de Educação PUC-Campinas. 2015; 20(1): 27–39. Publisher Full Text\n\nDuque P, Cervantes-Cervantes LS: University Social Responsibility: a systematic review and a bibliometric análisis. Estudios Gerenciales. 2019; 35(153): 451–464. Publisher Full Text\n\nWigmore-Álvarez A, Ruiz-Lozano M: University social responsibility (USR) in the global context: An overview of literature. Bus Prof Ethics J. 2012; 31(3/4): 475–498. Publisher Full Text\n\nAmiano Bonatxea I, Gutiérrez-Goiria J, Vazquez-De Francisco MJ, et al.: Is the global reporting initiative suitable to account for university social responsibility? Evidence from European institutions. International Journal of Sustainability in Higher Education. 2022; 23(4): 831–847. Publisher Full Text\n\nPlungpongpan J, Tiangsoongnern L, Speece M: University social responsibility and brand image of private universities in Bangkok. International journal of educational management. 2016; 30(4). Publisher Full Text\n\nMeseguer-Sánchez V, Abad-Segura E, Belmonte-Ureña LJ, et al.: Examining the research evolution on the socio-economic and environmental dimensions on university social responsibility. Int. J. Environ. Res. Public Health. 2020; 17(13): 4729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLatif KF: The development and validation of stakeholder-based scale for measuring university social responsibility (USR). Soc. Indic. Res. 2018; 140(2): 511–547. Publisher Full Text\n\nGomez L: The importance of university social responsibility in Hispanic America: A responsible trend in developing countries. Corporate social responsibility and sustainability: Emerging trends in developing economies. Emerald Group Publishing Limited; 2014; p. 241–68.\n\nGarzon Jimenez R, Zorio-Grima A: Sustainability engagement in Latin America firms and cost of equity. Academia Revista Latinoamericana de Administración. 2021; 34(2): 224–243. Publisher Full Text\n\nUrrunaga-Pastor D: Search strategy. figshare. Online resource.2023.\n\nSantos ME, Villatoro P: A multidimensional poverty index for Latin America. Review of Income and Wealth. 2018; 64(1): 52–82. Publisher Full Text\n\nSalmi J, D’Addio A: Policies for achieving inclusion in higher education. Policy Reviews in Higher Education. 2021; 5(1): 47–72. Publisher Full Text\n\nBargain O, Aminjonov U: Poverty and covid-19 in africa and latin america. World Dev. 2021; 142: 105422. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRababah A, Nikitina NI, Grebennikova VM, et al.: University social responsibility during the COVID-19 pandemic: Universities’ case in the BRICS countries. Sustainability. 2021; 13(13): 7035. Publisher Full Text\n\nLemos Lourenço M, Rosalia Ribeiro Silva M, Santana Galvão Oliveira R: University social responsibility and empathy in organizations during COVID-19 pandemic in Brazil. Social Responsibility Journal. 2022; 18(4): 806–824. Publisher Full Text\n\nAdel HM, Zeinhom GA, Younis RAA: From university social-responsibility to social-innovation strategy for quality accreditation and sustainable competitive advantage during COVID-19 pandemic. Journal of Humanities and Applied Social Sciences. 2022; 4(5): 410–437. Publisher Full Text\n\nGonzález JLL, Castro ARS, Mesa MLC, et al.: Scientific production in latin america and the caribbean in the period 1996-2019. Revista Cubana de Medicina Militar. 2020; 49(3).\n\nCarvajal-Tapia AE, Carvajal-Rodríguez E: Status of scientific production in Medicine in South America. 1996-2016. Revista de la Facultad de Medicina. 2018; 66(4): 595–600. Publisher Full Text\n\nHuamani C, González AG, Curioso WH, et al.: Scientific production in clinical medicine and international collaboration networks in South American countries. Rev Med Chil. 2012; 140(4): 466–475. PubMed Abstract \| Publisher Full Text\n\nHernández-Vásquez A, Bendezu-Quispe G, Comandé D, et al.: Worldwide original research production on maternal near-miss: A 10-year bibliometric study. Revista Brasileira de Ginecologia e Obstetrícia. 2020; 42: 614–620. PubMed Abstract \| Publisher Full Text\n\nMaina-Ahlberg B, Nordberg E, Tomson G: North-South health research collaboration: challenges in institutional interaction. Soc Sci Med. 1997; 44(8): 1229–1238. PubMed Abstract \| Publisher Full Text\n\nChandiwana S, Ornbjerg N: Review of North-South and South-South cooperation and conditions necessary to sustain research capability in developing countries. J. Health Popul. Nutr. 2003; 288–297.\n\nDuque P, Cervantes-Cervantes LS: University Social Responsibility: a systematic review and a bibliometric análisis. Estudios Gerenciales. 2019; 35(153): 451–464. Publisher Full Text\n\nQuienes Somos\|URSULA.[cited 2023 Jul 15]. Reference Source\n\nCasanueva-Yáñez G, Cantillo-Orozco AS, Maldonado-Córdova C, et al.: Social Responsibility Of Latin American Teachers Within The Framework Of Quality Education From The 2030 Agenda And The Sustainable Development Goals Of The United Nations. Journal of Positive School Psychology. 2022; 6(8): 2223–2235.\n\nUrrunaga-Pastor D: Database. Dataset. figshare. 2023." }	[ { "id": "217335", "date": "13 Nov 2023", "name": "Maria Jesus Martinez Usarralde", "expertise": [ "Reviewer Expertise Comparative Education. International Education. Teaching Education. Service 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\nThe article needs some improvement:\nThe introduction is excessively brief, as is the conclusion.\n\nIn the \"search strategy\" section, they point out that they have restricted language and year, but the Vosviewer programme only codes articles in English.\n\n\"There are no significant differences between high and low income countries\". This is not being evidenced and is based on a study that has nothing to do with the research field.\n\nParagraph on co-occurrence: the desire expressed by the authors cannot be derived from a co-occurrence analysis.\n\n\"The bibliometric analysis is the first to evaluate the scientific production of USR associated with Latin American institutions\": the analysis referred to in the article is not observed in this article, in the sense that the data do not reflect the scientific production of USR in Latin American institutions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "11953", "date": "18 Jul 2024", "name": "Diego Urrunaga-Pastor", "role": "Author Response", "response": "Dear Reviewers, We appreciate your review of our manuscript and the comments you provided. Below, we have addressed each of your observations in detail: Comment 1: The introduction is excessively brief, as is the conclusion. Response: We appreciate your comment. Relevant Information was added to the manuscript regarding bibliometric analysis. Comment 2: In the \"search strategy\" section, they point out that they have restricted language and year, but the Vosviewer programme only codes articles in English. Response: We appreciate your comment. The document states, “The search was not restricted by year or language of publication.” No change was conducted. Comment 3: \"There are no significant differences between high- and low-income countries\". This is not being evidenced and is based on a study that has nothing to do with the research field. Response: We appreciate your comment. The redaction was improved. The writing was improved according to the comment. Comment 4: Paragraph on co-occurrence: the desire expressed by the authors cannot be derived from a co-occurrence analysis. Response: Thank you for the recommendation. Regarding the co-occurrence overlay visualization, the attribute average publication year of the document in which a keyword or a term occurs was used in the document. As it is described in the VOSViewer manual about this attribute, “The average publication year of the documents in which a keyword or a term occurs or the average publication year of the documents published by a source, an author, an organization, or a country.” Hence, the interpretation of this visualization is appropriate for discussion. Information was added in methods to clarify. Comment 5: \"The bibliometric analysis is the first to evaluate the scientific production of USR associated with Latin American institutions\": the analysis referred to in the article is not observed in this article, in the sense that the data do not reflect the scientific production of USR in Latin American institutions. Response: We appreciate your comment. The expression “associated with” was replaced with “from” to be more precise as it is stated in the reviewer comment." } ] }, { "id": "225262", "date": "14 Feb 2024", "name": "Mikel Perez-Gutierrez", "expertise": [ "Reviewer Expertise Bibliometrics", "Sport Sciences", "Physical 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 manuscript carried out a bibliometric analysis of the scientific production indexed in Scopus focused on University Social Responsibility by Latin America and Caribbean institutions. The manuscript offers an overview of the main bibliometric indicators for characterising this scientific output. However, some deficiencies should be amended for improving the quality and reproducibility of the research (methodology section) as well as the understanding of the obtained results (results, discussion and conclusions sections). These suggestions are the following:\nPage 3: For improving the understanding of the manuscript and its object of study, Latin America and the Caribbean countries should be defined and justified. For that purpose, CELAC (Comunidad de Estados Latinoamericanos y Caribeños) could be used for defining the countries considered under the term \"Latin America and the Caribbe\". For citation, these pages should be consulted and cited: http://s017.sela.org/celac/quienes-somos/que-es-la-celac/ or https://es.wikipedia.org/wiki/Comunidad_de_Estados_Latinoamericanos_y_Caribe%C3%B1os), Page 3: A justification of the database chosen for data retrieval should be included. The work carried out by Gusenbauer and Haddaway (2020) analysed the coverage and breadth of several databases, concluding Scopus is a principal database and justifying its use in the present manuscript. (Gusenbauer et al) 1 Page 3: All bibliometric indicators included in the results section should be explained. Total citations and the average citations per article by each country are included in Table 2. Table 3 presents the number of articles published in the most important journals. These indicators should be mentioned here. Page 3: According to the results presented in Figure 4, an explanation about the analysis applied for the temporal evolution of the term co-occurrence and the presentation of results (temporal evolution by means of colours?) should be included. How this temporal evolution is presented in Figure 4? Page 4: The difference between \"author\" and \"mention\" should be explained in the methodology section. Are \"mentions\" referring to \"signatures\"? Page 4: Regarding the obtained results presented in Table 2, the highest average of citations per article was obtained by Lebanon. If authors are referring to the Latin America and Caribbean countries with the highest average citations per article, Colombia achieved the highest (8.4), followed by Chile (5.4) and Brazil (4.1). The sentence should be amended for clarifying this information. Page 5: The title of Table 1 (Authors with the highest scientific production) is not representing the results included since there are three authors with one paper, but there are more authors with only one paper. The title of the table and the results included should be amended. Firstly, authors with 2 or more papers should be only included in Table 1. Thus, the title of Table 1 should be the following: \"Authors with two or more articles indexed in Scopus...\" Page 5: Table 2 should also include the amount of papers by country for comparing their productivity. Moreover, in the results section the percentage of publications per country is described (Brazil 26.4%, Chile 17% and Colombia 13.2%). Page 6: As it was mentioned in Table 1, the title of Table 3 (Journals with the highest scientific production) is not representing the results included since there are three journals with one paper, but it seems there are more journals with only one paper. The title of the table and the results included should be amended. Firstly, journals with 3 or more papers should be only included in Table 3. Thus, the title of Table 3 should be the following: \"Journals with three or more publications...\" Page 7: The results obtained in the present manuscript should be compared with those obtained by Duque and Cervantes-Cervantes (2019) since their topic is the same. Specifically, results could be compared according to total number of articles focused on University Social Responsibility, distribution of articles by country, distribution of articles by year, most productive authors and most important journals for disseminating USR scientific output. This comparison is relevant for determining the contribution of Latin America and Caribbean countries, institutions or authors to the worldwide scientific output in this topic. Page 8: \"To the best of our knowledge, this bibliometric analysis is the first to evaluate the scientific production on USR associated with institutions in Latin America\". According to the bibliometric analysis developed, the present manuscript is evaluating the scientific production on USR published by Latin America and Caribbean institutions. The sentence should be amended. Page 8: The conclusion section should be strengthened. An explanation about the causes and consequences of this analysis should be included. Some questions to answer are: What is the relevance of Latin America and Caribbean institutions regarding USR scientific output? Who is the most important author/country? Is collaboration among authors, institutions or countries essential for developing research on USR? What are the strengths and weaknesses of this filed of knowledge? What knowledge gaps need to be filled? To which countries should investment in research be directed?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11954", "date": "18 Jul 2024", "name": "Diego Urrunaga-Pastor", "role": "Author Response", "response": "Dear Reviewer, We appreciate your review of our manuscript and the comments you provided. Below, we have addressed each of your observations in detail: Comment 1: Page 3: For improving the understanding of the manuscript and its object of study, Latin America and the Caribbean countries should be defined and justified. For that purpose, CELAC (Comunidad de Estados Latinoamericanos y Caribeños) could be used for defining the countries considered under the term \"Latin America and the Caribbean\". For citation, these pages should be consulted and cited: [http://s017.sela.org/celac/quienes-somos/que-es-la-celac/](http://s017.sela.org/celac/quienes-somos/que-es-la-celac/) or [https://es.wikipedia.org/wiki/Comunidad_de_Estados_Latinoamericanos_y_Caribe%C3%B1os](https://es.wikipedia.org/wiki/Comunidad_de_Estados_Latinoamericanos_y_Caribe%C3%B1os). Response: We appreciate your comment. All the document sections were reviewed and improved. Since the provided reference by the reviewer was not available, the Latin America & Caribbean countries list by United Nations was used. Comment 2: Page 3: A justification of the database chosen for data retrieval should be included. The work carried out by Gusenbauer and Haddaway (2020) analyzed the coverage and breadth of several databases, concluding Scopus is a principal database and justifying its use in the present manuscript. Response: We appreciate your comment. We cited documents in the methods to justify the database used (Scopus). Comment 3: Page 3: All bibliometric indicators included in the results section should be explained. Total citations and the average citations per article by each country are included in Table 2. Table 3 presents the number of articles published in the most important journals. These indicators should be mentioned here. Response: We appreciate your comment. Regarding the clarification of all bibliometric indicators, there are stated in the methods: “Additionally, useful indicators for bibliometric evaluation, such as the authorship rate (ratio between the total number of articles and the total number of authors), co-authorship rate (average number of co-authors per article), and collaboration rate (ratio between the total number of authors of articles with multiple authors and the total number of articles with multiple authors) were described.” The information about the results presented in Tables 2 and 3 was added to the methods. Comment 4: Page 3: According to the results presented in Figure 4, an explanation about the analysis applied for the temporal evolution of the term co-occurrence and the presentation of results (temporal evolution by means of colours?) should be included. How is this temporal evolution presented in Figure 4? Response: We appreciate your comment. In methods, information was added to clarify. Additionally, the figure 2 was reuploaded since in the reviewed manuscript, it seems that the legend (colors according to the timeline in years) was not presented. Comment 5: Page 4: The difference between \"author\" and \"mention\" should be explained in the methodology section. Are \"mentions\" referring to \"signatures\"? Response: We appreciate your comment. This information was confusing. We improve the redaction. Comment 6: Page 4: Regarding the obtained results presented in Table 2, the highest average of citations per article was obtained by Lebanon. If authors are referring to the Latin America and Caribbean countries with the highest average citations per article, Colombia achieved the highest (8.4), followed by Chile (5.4) and Brazil (4.1). The sentence should be amended for clarifying this information. Response: We appreciate your comment. This table was improved, and the information reported about this table. Comment 7: Page 5: The title of Table 1 (Authors with the highest scientific production) is not representing the results included since there are three authors with one paper, but there are more authors with only one paper. The title of the table and the results included should be amended. Firstly, authors with 2 or more papers should be only included in Table 1. Thus, the title of Table 1 should be the following: \"Authors with two or more articles indexed in Scopus...\" Response: We appreciate your comment. Table 1 was updated with the suggestions. Comment 8: Page 5: Table 2 should also include the amount of papers by country for comparing their productivity. Moreover, in the results section, the percentage of publications per country is described (Brazil 26.4%, Chile 17% and Colombia 13.2%). Response: We appreciate your comment. This table was improved, and the information reported about this table. Comment 9: Page 6: As it was mentioned in Table 1, the title of Table 3 (Journals with the highest scientific production) is not representing the results included since there are three journals with one paper, but it seems there are more journals with only one paper. The title of the table and the results included should be amended. Firstly, journals with 3 or more papers should be only included in Table 3. Thus, the title of Table 3 should be the following: \"Journals with three or more publications...\" Response: We appreciate your comment. The table was updated with the suggestion. Comment 10: Page 7: The results obtained in the present manuscript should be compared with those obtained by Duque and Cervantes-Cervantes (2019) since their topic is the same. Specifically, results could be compared according to total number of articles focused on University Social Responsibility, distribution of articles by country, distribution of articles by year, most productive authors and most important journals for disseminating USR scientific output. This comparison is relevant for determining the contribution of Latin America and Caribbean countries, institutions or authors to the worldwide scientific output in this topic. Response: We appreciate your comment. We added the recommended document in the discussion of the results. Comment 11: Page 8: \"To the best of our knowledge, this bibliometric analysis is the first to evaluate the scientific production on USR associated with institutions in Latin America\". According to the bibliometric analysis developed, the present manuscript is evaluating the scientific production on USR published by Latin America and Caribbean institutions. The sentence should be amended. Response: We appreciate your comment. The sentence was amended. Comment 12: Page 8: The conclusion section should be strengthened. An explanation about the causes and consequences of this analysis should be included. Some questions to answer are: What is the relevance of Latin America and Caribbean institutions regarding USR scientific output? Who is the most important author/country? Is collaboration among authors, institutions or countries essential for developing research on USR? What are the strengths and weaknesses of this field of knowledge? What knowledge gaps need to be filled? To which countries should investment in research be directed? Response: We appreciate your comment. The conclusion was improved" } ] } ]	1	https://f1000research.com/articles/12-1340
https://f1000research.com/articles/13-107/v1	16 Feb 24	{ "type": "Research Article", "title": "Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38", "authors": [ "Zamroni Afif", "Mochammad Istiadjid Eddy Santoso", ". Nurdiana", "Husnul Khotimah", "Irawan Satriotomo", "Shahdevi Nandar Kurniawan", "Hidayat Sujuti", "Dheka Sapti Iskandar", "Annisatul Hakimah", "Mochammad Istiadjid Eddy Santoso", ". Nurdiana", "Husnul Khotimah", "Irawan Satriotomo", "Shahdevi Nandar Kurniawan", "Hidayat Sujuti", "Dheka Sapti Iskandar" ], "abstract": "Background: Insomnia is difficulty initiating or maintaining sleep for at least three nights a week or more and lasting for at least 3 months. One of the molecules that play a role in the circadian rhythm of arousal system is hypocretin/orexin. Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. Centella asiatica (CA) has a role in the signal work of the MAPK/ERK, Akt, and p38 path in many various diseases.\nMethods: The research method used is true laboratory experimental. The research approach used was randomized control group post-test only. Zebrafish embryos aged 0-7 dpf were used in this study. The treatment group consisted of 5 groups: normal, insomnia, insomnia + 2.5 μg/mL CA, insomnia + 5 μg/mL CA, and insomnia + 10 μg/mL CA. The locomotor motion of zebrafish larvae was observed using Basler cameras on days five-, six- and seven-day post fertilization (dpf), then analyzed by using Western Blot method.\nResults: The results proved that exposure to CA extract was able to reduce the expression of orexin (91963 ± 9129) and p38 (117425 ± 6398) as an arousal trigger in the sleep-wake cycle, with the most optimal concentration of CA 5 μg/mL. Exposure to CA extract was also able to reduce the expression of ERK (94795 ± 30830) and Akt (60113.5 ± 27833.5) with an optimum concentration of CA 2.5 μg/mL.\nConclusion: Exposure to CA extract was able to improve the sleep activity of zebrafish larvae insomnia model by extending the total inactivity time (cumulative duration) and shortening the duration of first sleep (latency to first) in light and dark phases through inhibition of orexin, ERK, p38, and Akt.", "keywords": [ "Insomnia", "Centella asiatica", "Orexin", "ERK", "p38", "Akt." ], "content": "Introduction\n\nThe 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and the third edition of the International Classification of Sleep Disorders (ICSD-3) define insomnia as difficulty initiating or maintaining sleep at least 3 nights a week or more and lasting at least 3 months.1 The prevalence in Europe and Scandinavia shows an increasing prevalence. British study of more than 20,000 adults saw an increase in the average prevalence of insomnia over a 15-year period from 35% in 1993 to 38.6% in 2007, while the prevalence of insomnia in the United States was about 27%.1,2\n\nBased on gender, insomnia is more common in women than in men, which is 50% greater in women than men. Old age, low economic status, hyperarousal conditions, and the presence of psychiatric or medical comorbidities are risk factors associated with insomnia.1 The latest concept of insomnia is the disintegration of molecules that play a role in alternation of waking and sleeping rhythms in the brain. One of the molecules that play a role in the circadian rhythm of arousal is hypocretin/orexin and histamine, while those that play a role in sleep, such as γ-aminobutyric acid (GABA), adenosine, serotonin, melatonin, and prostaglandin D2 (PGD2).3\n\nThere are 2 receptor subtypes for orexin that will activate 3 G-protein subtypes (Gq/11, Gi/10, and Gs). Activation of Orexin Receptor-1 (OXR1 or OA) and Orexin Receptor-2 (OXR2 or OB) receptors activates phospholipase c cascade (PLC-IP3/DAG). Orexin receptor signaling pathways phospholipase D (PLD)/phosphatidic acid (PA), phospholipase A (PLA)/arachidonic acid (AA), and mitogen-activated protein kinase cascade (MAPK). Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. Extracellular Signal-regulated Kinases or ERK1/2 and p38 are rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly the Gi pathway. Orexin binding and orexin receptors will activate intracellular calcium PKC-dependent or independent pathways. Orexin receptors, both OA and OB, also stimulated the activation of Akt kinase in the cortical nerves of hypoxic stress-stressed rats. In addition, orexin will cause a reaction on OB to activate Gi proteins causing inhibition of cAMP formation. Orexin also activates OA to stimulate cAMP synthesis. Orexin signaling rapidly activates the mTORC1 pathway triggered by the lysosomal v-ATPase pathway.4\n\nCentella asiatica has a role in the signal work of the MAPK/ERK pathway. Studies on human breast cancer cells show that Asiatic acid (AA) in gotu kola plays a role in inducing inhibition of cancer cell growth through mediators such as MAPK, ERK1/2, and p38. Asiatic acid targets P13K/Akt/mTOR to prevent growth and induction of apoptosis in ovarian cancer cells. In cardiovascular disease AA has benefits with its ability to inhibit excessive pressure that induces cardiac hypertrophy by inhibition of phosphorylation of p38, MAPK, and ERK1/2 and decreases the production of TGF-β and NF-КB.5,6\n\nCurrently, many animal models of sleep have been developed for sleep disorder research. The zebrafish (Danio rerio) currently exhibit vertebral sleep models that are important among mouse and invertebrate models. Zebrafish sleep mainly at night as in humans, unlike rats that have the habit of sleeping during the day. The main advantages of zebrafish as sleep model animals are the discovery of genes involved in sleep control, neuropeptide systems, transparent anatomical structures of the brain, and allow for neuropsychiatric disease models, as well as models for genetic and pharmacological screening in sleep disorders.7 Zebrafish brains also contain orexin, one of the molecules important for sleep regulation in mammalian brains. At present high-speed infrared video capture combined with computational image analysis has been used to quantitatively describe locomotor behavior and sleep posture in zebrafish larvae.3 The purpose of this study was to prove the effect of gotu kola extract (Centella asiatica) in improving the sleep activity of zebrafish larvae (Danio rerio) insomnia model, extending the total inactivity time (cumulative duration) and shortening the duration of first sleep (latency to first) in light and dark phases through inhibition of orexin, ERK, p38 and Akt.\n\n\nMethods\n\nThe research design used in this study is a true laboratory experiment. The research approach used is randomized control group posttest only.\n\nMaintenance of zebrafish embryos with exposure of dark:bright for 12:12 hours and plankton feeding are given three times a day, conductivity 350-600 S and salinity 0-0.6 ppt (parts per thousand). Zebrafish embryos aged three–seven-day post fertilization (dpf) was used in this study, obtained from the Reproductive Laboratory of the Faculty of Fisheries and Marine Sciences, Universitas Brawijaya.\n\nThis study used as many as three pieces of 48 well plates in each treatment group. The total number of embryos needed in this study was 144 embryos per group, so that the total number of zebrafish embryos included in the study in all treatment groups was 720 embryos. The Faculty of Medicine Health Research Ethics Committee Universitas Brawijaya has given its approval for this project. Ethics License No: 147/EC/KEPK-S3/06/2021.\n\nThe inclusion criteria in this study were healthy, clear embryos, not contaminated with fungi or parasites. Embryo hatching 2-3 dpf (day post fertilization). While the exclusion criteria are zebrafish larvae that died or were deformed during the study.\n\nThe light treatment used in this study to cause insomnia conditions in zebrafish larvae refers to the research method of Pinheiro-da-silva et al. (2016) with modification of light exposure as follows: negative control group with light exposure 12 hours light and 12 hours dark (normal group), and positive control group with light treatment 24 hours light (insomnia group).8 Light is given at 200 lux from 0 dpf until the age of 7 dpf. Modifications were made to the design of lamps and tools used at the time of the study.9\n\nFish larvae movements were analyzed with video recordings using Ethovision XT software. The zebrafish larvae are sleep when the zebrafish is immobile for one minute. Sleep latency is the period when the lights are turned off and the first sleep period appears. Zebrafish larvae experience insomnia when the sleep latency is more than 20 minutes.9\n\nThe administration of Centella asiatica ethanol extract with concentrations of 2.5 μg/ml, 5 μg/ml, and 10 μg/ml (Khotimah et al., 2015), sequentially introduced into the treatment group with 24 hours of light exposure group. Centella asiatica extract is given once in 24 hours at the same time as the media is replaced.\n\nZebrafish larva samples were lyzed in a suitable 5x RIPA buffer volume [50 mM Tris-Cl pH 7.4, 750 mM NaCl, 0.5% Sodium Deoxycholate, 0.5% SDS, 5% Triton, 1 mM Sodium Orthovanadate, 50 mM NaF, 5 μg/ml Pepstatin, 5 μg/ml Aprotinin, 5 μg/ml Leupeptin] on ice for 30 min followed by centrifugation at 14000 g for ten min. One volume of 100% (w/v) TCA was added to four sample volumes and incubated for ten min at 4°C. After centrifugation at 14000 rpm for five minutes, the pellets are removed and washed twice with cold acetone. The pellets are heated to 95°C for five-ten minutes and mixed in 50 μl urea 6 M in 50 mM bicarbonate. Protein content was assessed using a micro-BCA assay. Five μL of sample was added to the 96-well microplate followed by 100 μL of BCA reagent. The plates were incubated in the dark for 30 min at 37°C and absorbance was measured at 560 nm and protein concentration was determined from the BSA standard curve.10\n\nEach sample consisted of six zebrafish larvae aged seven days. All cell extracts were prepared in suspension in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40 and 0.5% sodium deoxycholate) with protease inhibitors. The protein sample was separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride membrane. The membrane is blocked with 5% fat-free milk then the membrane was probed with primary antibodies overnight at 4°, followed by incubation with HRP-conjugated secondary antibodies for 1 hour. The blot is formed using an ECL kit according to the manufacturer’s instructions. Densitometric analysis was demonstrated using QuantityOne software (Bio-Rad).5\n\nAll data were analyzed using SPSS 25, a parametric test. Data that were normally distributed and homogeneous were analyzed using One-way ANOVA statistical analysis to determine the difference between the treatment groups provided. If the p-value < 0.05, it was considered statistically significant. Results expressed in mean ± SD.\n\n\nResults\n\nProtein expression of orexin was measured in zebrafish larvae samples using the western blot method and gel doc chemiluminescence. The average results of orexin protein WB analysis are presented in the form of tables and diagrams as follows (Table 1 and Figure 1).\n\n\n\n1. Normal Group (N) = kontrol negatif\n\n2. Insomnia Group (I) = induksi cahaya\n\n3. I + 2.5 μg/mL CA\n\n4. I + 5 μg/mL CA\n\n5. I + 10 μg/mL CA\n\n6. Marker (Orexin)\n\nBased on the average results of WB analysis of orexin protein expression, the insomnia group has a higher average expression value of 131239 ± 7994 compared to the normal group (122716.5 ± 5014.5) and different concentrations of CA extract which are respectively 2.5 μg/mL (100565 ± 9707), 5 μg/mL (91963 ± 9129) and 10 μg/mL (92655 ± 9604). Various concentrations of CA extract can reduce the average expression value of orexin protein which basically functions as a wake-up trigger in the sleep-wake cycle. Statistical analysis also showed that there was a significant difference in orexin protein expression values, this was evidenced by significance signs in the form of (a,b,c) which showed that exposure treatment of CA extract was proven to reduce orexin protein expression values significantly lower than normal and insomnia groups. The location of orexin protein is at 49 kDa (Table 1 and Figure 1).\n\nExtracellular Signal Regulated Kinase (ERK) protein expression was measured in zebrafish larvae samples using the western blot method and gel doc chemiluminescence. The average results of WB analysis of ERK protein are presented in the form of tables and diagrams as follows (Table 2 and Figure 2).\n\nBased on the average results of WB analysis of ERK protein expression, the normal and insomnia groups have higher average expression values of 285461.5 ± 22617.5 and 211092.5 ± 36867.5 compared to different CA extract concentration treatment groups, namely 2.5 μg/mL (94795 ± 30830), 5 μg/mL (133577 ± 44730), and 10 μg/mL (110930.5 ± 40407.5), respectively. Various concentrations of CA extract were able to reduce the average expression value of ERK protein which functions in the mechanism of orexin protein activation as a wake-up trigger in the sleep-wake cycle. Statistical analysis also showed that there was a significant difference in the value of ERK protein expression, this was evidenced by signs of significance in the form of (a,b,c) which showed that exposure treatment of CA extract was proven to reduce the value of ERK protein expression significantly lower than normal and insomnia groups. The location of the ERK protein is 42 kDa (Table 2 and Figure 2).\n\nAKT Protein expression was measured in zebrafish larvae samples using western blot and gel Doc chemiluminescence methods. The average results of the AKT protein WB analysis are presented in the form of tables and diagrams as follows (Table 3 and Figure 3).\n\nBased on the average results of WB analysis, Akt protein expression can be seen that the normal group and exposure treatment of CA extract concentration of 5 μg/mL have higher average expression values of 181687.5 ± 69844.5 and 120525 ± 50892 compared to the insomnia group (70468.5 ± 34160.5) and treatment of CA extract concentration of 2.5 μg/mL (60113.5 ± 27833.5) and 10 μg/mL (79935.5 ± 29777.5). The concentration of CA extract 2.5 μg/mL was able to reduce the average Akt protein expression value lower than the insomnia group. Statistical analysis shows that there is a difference in the value of Akt protein expression this is evidenced by the value of p-value = 0.045 (<0.05) which means there is a significant difference, but in post-hoc follow-up tests show that the sign of significance is in the form of (a) all which means that the average value of Akt expression for each group has no real difference. Therefore, from these results it can be concluded that the treatment of CA extract with the most optimal concentration of 2.5 μg/mL can improve sleep activity in zebrafish larvae insomnia models through decreased Akt protein expression. The location of the Akt protein is 60 kDa (Table 3 and Figure 3).\n\np38 Protein expression was measured in zebrafish larvae samples using western blot method and gel doc chemiluminescence. The average results of WB protein p38 analysis are presented in the form of tables and diagrams as follows (Table 4 and Figure 4).\n\nBased on the average results of WB analysis of p38 protein expression, it can be seen that the normal group and the treatment of exposure to CA extract concentration of 10 μg/mL have a higher average expression value of 194368.5 ± 20149.5 and 158576 ± 23533 compared to the insomnia group (146856.5 ± 21715.5) and the treatment of CA extract concentration of 2.5 μg/mL (123814 ± 9) and 5 μg/mL (117425 ± 6398). The concentration of CA extract of 2.5 μg/mL and 5 μg/mL was able to reduce the average p38 protein expression value lower than the insomnia group. The p38 protein functions in the mechanism of orexin protein activation that can affect the sleep-wake cycle. Statistical analysis shows that there is a significant difference in the expression value of the p38 protein, this is evidenced by the value of p-value = 0.002 or <0.05 which means there is a significant difference. The location of the p38 protein is 38 kDa (Table 4 and Figure 4).\n\n\n\na. Light phase\n\nThe following are the results of fish motion analysis of total inactivity time (cumulative duration) zebrafish larvae in the normal group, insomnia group (24-hour light exposure), and the group with the administration of CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during the light phase (Table 5 and Figure 5).\n\nb. Dark phase\n\nThe following are the results of fish motion analysis of total inactivity time (cumulative duration) zebrafish larvae normal group, insomnia group (24-hour light exposure), and group with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during the dark phase (Table 6 and Figure 6).\n\nBased on the results of observations of locomotor motion of zebrafish larvae in normal groups, insomnia groups (24-hour light exposure), and groups with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during light and dark phases, it can be concluded that exposure to CA extract with different concentrations can affect the total inactivity time (cumulative duration) in zebrafish larvae to be longer than the insomnia group (Tables 5, 6 and Figures 5, 6).\n\nBased on the results of observations of locomotor motion of zebrafish larvae in normal groups, insomnia groups (24-hour light exposure), and groups with CA extract concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL during light and dark phases, it can be concluded that exposure to CA extract with different concentrations can affect the average total time of first sleep (latency to first) in zebrafish larvae to be shorter than the insomnia group. This can be evidence that exposure to CA extract can improve insomnia conditions in zebrafish larvae by shortening the first time of sleep (latency to first) (Tables 7, 8 and Figures 7, 8).\n\n\nDiscussion\n\nInsomnia is primarily characterized by individual dissatisfaction with the duration or quality of sleep accompanied by functional impairment during the day. A person with insomnia usually experiences one or more symptoms such as fatigue, lack of energy, difficulty concentrating, and mood disorders. Insomnia can appear as a major complaint or more often occur along with other medical or psychiatric disorders, such as pain disorders and depression.11 A 30-minute rule is used to meet insomnia criteria, including taking >30 minutes to fall asleep, spending >30 minutes awakening after sleep onset or waking >30 minutes before the desired time and before reaching 6.5 hours of sleep.12\n\nThe latest concept of insomnia suggests the disintegration of molecules that play a role in alternation of wake and sleep rhythms in the brain. Molecules that play a role in the circadian rhythm of waking up are catecholamines, orexin, and histamine, while those that play a role for sleep, such as γ-aminobutyric acid (GABA), adenosine, serotonin, melatonin, and prostaglandin D2 (PGD2).7\n\nHypocretin (Hcrt) (also known as orexin) is a highly conserved prepro-Hcrt peptide product, with 2 enzymatically cleaved Hcrt peptides: Hcrt 1 or orexin A (33 amino acids) and Hcrt 2 or orexin B (28 amino acids). These peptides are synthesized in a group of neurons in the lateral hypothalamus (Hypo), and their neuronal processes extend widely in the brain. Mammals have 2 Hcrt receptors (HcrtRs) (HcrtR1/orexin A/OA receptor and HcrtR2/orexin B/OB receptor) that are distributed throughout the central nervous system and in peripheral organs. To date, a large amount of evidence suggests that Hcrt is involved in various physiological processes, such as sleep/wakefulness, food intake, and energy homeostasis. The HCRT system has also been reported to regulate reproduction.13 Other studies have reported that in zebrafish, as in mammals, orexin signaling is involved in the regulation of many physiological functions, such as sleep/wake cycles, energy homeostasis, and locomotor activity.14\n\nHypocretin (Hcrt) or orexin of the nearby hypothalamus synthesize and secrete Hcrts or orexin in the hypothalamus and are recognized primarily as important regulators of sleep or wakefulness, energy homeostasis, and appetite. Other researchers have previously shown that excessive orexin expression can cause insomnia-like phenotypes in zebrafish and that treatments such as orexin inhibition can stimulate feeding behavior in zebrafish. The effects of orexin on zebrafish sleeping and eating activity are very similar to what has been reported in mammals.13\n\nThe main elements involved in the wake mechanism are orexin or hypocretinergic and histaminergic from the posterior portion of the lateral hypothalamus. Activated orexin will increase phosphorylated ERK1/2 levels. ERK1/2 is rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly via Gi. In addition, dopamine and histamine cell groups have the highest density of hypocretin-2 receptors or OB.1 Activated H1R (histamine-1 receptor) will stimulate immune responses Th1 and Th2. Histamine H1 receptors are also expressed in skin dendrite cells and keratinocytes in skin tissue, and histamine increases NGF production over human keratinocyte H1R. NGF secretion is due to phosphorylation of protein kinase C, extracelullar signal regulated kinase (ERK), and activation of AP-1 as a result of H1 stimulation.15\n\nAnother study explained that orexin, both OA and OB, also stimulated the activation of Akt kinase in the cortical nerves of hypoxic stress-stressed rats. In addition, Akt kinase also plays a role in several signaling pathways for apoptosis induction and ovarian cancer.3 Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. ERK1/2 and p38 are rapidly phosphorylated in response to OA and OB, mediated primarily by Gq and slightly through the Gi pathway.3\n\nCentella asiatica acts as a neuroprotector against various neurological disorders. The promising potential of Centella asiatica against neurological disorders can be attributed to its antioxidant, anti-inflammatory, anxiolytic, and stress-fighting properties. Centella asiatica in rats created with sleep deprivation for 72 hours significantly improved locomotor activity, anti-anxiety effects, lowered cortisol levels as well as improved neuronal inflammation, and apoptosis response.16 Centella asiatica provides a calming effect on activity in experimental animals, can increase phenobarbitone which has the effect of inducing sleep time and reducing immobility in experimental animals.17\n\nResearch by Kumar illustrates those 8 days of Centella asiatica treatment provides no benefit for inducing sleep but still shows a protective effect against sleep-induced anxiety (sleep deprivation). The neuroprotective effects of Centella asiatica in sleep deprivation conditions that induce anxiety-like behaviors and the anxiolytic effects of Centella asiatica are modulated by NO (nitric oxide).16\n\nAnother study reported that one of the active compounds of Centella asiatica extract, Asiatic Acid (AA), showed an important role against lipopolysaccharide (LPS)/d-GaIN-induced fulminant hepatic failure with inhibition of oxidative stress and inflammation. The proposed mechanism is related to inhibition in MAPK and NF-КB. This study showed that AA induces anti-inflammatory effects through the activation of antioxidant enzymes by suppressing CCI and MAPK activity (p38, ERK1/2, and JNK, as well as increasing Nrf2 activity).4\n\nThe effect of one of the active substances of Centella asiatica, namely Asiatic acid (AA) on P13 kinase (P13K)/Akt/mTOR has also been investigated as a therapeutic target in ovarian cancer cells. Phosphorylation of P13K, Akt, and mTOR decreased in AA-treated cells, indicating inactivation of the P13K/Akt/mTOR pathway by AA. Asiatic acid has a target at P13K/Akt/mTOR to prevent growth and induction of apoptosis in ovarian cancer cells.5 Another study on the benefits of Centella asiatica against inflammatory reactions is mainly played by Centella asiatica compounds, showed that AA protects human bronchial cells against oxidative and inflammatory damage through mitochondrial stability, decreases ROS and PGE2 production, and suppresses the expression of NADPH oxidase proteins, COX-2, NF-КB p65, and p-p38.5\n\nCumulative duration is the total length of time the fish is asleep or in an inactive condition. The characteristics of zebrafish when asleep are that adult zebrafish stop swimming for 6 seconds, and zebrafish larvae stop for 1 minute, do not move at the bottom or surface, and are less sensitive to external stimuli. Zebrafish larval activity is very minimal at night.18 Zebrafish have cells that are responsive to light19 and the duration of exposure to light received by fish leads to a decrease in melatonin production which produces several inputs that promote excessive wakefulness in the brain.19\n\nSleep latency or latency to first is defined as the time (both day and night) needed to cause the first sleep.9 In this study, it was found that the lengthening of latency to first time or sleep latency in zebrafish larvae models exposed to 24-hour light. Continuous exposure to light can cause this condition, according to the theory that zebrafish have endogenously controlled circadian rhythm behaviors that can be affected by light. In another study obtained with a treatment of 14 hours:10 hours of light: dark, it was found that zebrafish larval activity occurred maximally in the phase of the lights turned on. From this study, the average sleep latency at light ranged from 24.8 to 59.2 minutes on the 6th and 7th day experiments with light exposure during the day.9 The regulation of light to the sleep-wake cycle is related to the regulation of melatonin and the hypocretin/orexin system (Hcrtr). As much as 60% of these latency conditions decrease at night, which indicates the formation of hcrtr.19\n\n\nConclusions\n\nBased on this study, it can be concluded that the effect of gotu kola extract (Centella asiatica) in improving the sleep activity of zebrafish larvae (Danio rerio) insomnia model by extending the total inactivity time (cumulative duration) and shortening the duration of the first time of sleep (latency to first) in light and dark phases through inhibition of orexin, ERK, p38 and Akt.", "appendix": "Data availability\n\nFigshare. data supporting research results Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38, DOI: 10.6084/m9.figshare.24078519. 20\n\nThe project contains the following basic data:\n\n• Graph of exposure light and concentration of Centella asiatica extract. xlsx\n\n• Graph of WB marker Orexin, ERK, AKT and P38.xlsx\n\n• Results of Statistical Analysis of cumulative duration light and dark phase.docx\n\n• Results of Statistical Analysis of latency to first light and dark phase.docx\n\n• Results of Statistical Analysis of WB AKT, ERK, Orexyn, and p38.docx\n\n• Figure of Result Western Blot Marker Orexin, ERK, AKT and P38.docx\n\nData is available under the terms of the Creative Commons Attribution 4.0 International (CC-BY 4.0) license.\n\nFigshare: The ARRIVE Essential 10: author checklist - Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38, DOI: 10.6084/m9.figshare.24078519. 20\n\nhttps://figshare.com/articles/dataset/preview_ARRIVE_10_checklist_author/24079407 21\n\n• The ARRIVE Essential 10: author checklist.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thanks to Faculty of Medicine Universitas Brawijaya Ministry of Education, Culture, Research, and Technology in accordance with the Universitas Brawijaya Directorate of Research and Community Service Program for funding this research. The author also would like to thank Prof. Dr. dr. Moch. Istiadjid Eddy Santoso, Sp.S, Sp.BS(K), M.Hum for his suggestions during the research.\n\n\nReferences\n\nLichstein KL, Taylor DJ, McCrae CS, et al.: Insomnia: Epidemiology and Risk Factors. Princ. Pract. Sleep Med. Fifth Ed. 2010; 827–837. Publisher Full Text\n\nLombardero A, Hansen CD, Richie AE, et al.: A Narrative Review of the Literature on Insufficient Sleep, Insomnia, and Health Correlates in American Indian/Alaska Native Populations. J. Environ. Public Health. 2019; 2019: 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiu CN, Prober DA: Regulation of zebrafish sleep and arousal states: current and prospective approaches. Front. Neural Circuits. Mar. 2013; 7(MAR). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, et al.: The Orexin/Receptor System: Molecular Mechanism and Therapeutic Potential for Neurological Diseases. Front. Mol. Neurosci. Jun. 2018; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLv J, Sharma A, Zhang T, et al.: Pharmacological Review on Asiatic Acid and Its Derivatives: A Potential Compound. SLAS Technol. Apr. 2018; 23(2): 111–127. PubMed Abstract \| Publisher Full Text\n\nRen L, Cao QX, Zhai FR, et al.: Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling. Pharm. Biol. Nov. 2016; 54(11): 2377–2382. PubMed Abstract \| Publisher Full Text\n\nBringmann H: Genetic sleep deprivation: using sleep mutants to study sleep functions. EMBO Rep. 2019; 20(3): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinheiro-da-Silva J, Silva PF, Nogueira MB, et al.: Sleep deprivation effects on object discrimination task in zebrafish (Danio rerio). Anim. Cogn. 2017; 20(2): 159–169. PubMed Abstract \| Publisher Full Text\n\nAfif Z, et al.: Light Exposure’S Effects on Inactive State Duration and Sleep Latency in Zebrafish (Danio Rerio) Larvae Insomnia Model. MNJ (Malang Neurol. Journal). 2022; 8(2): 129–134. Publisher Full Text\n\nBrennan K, et al.: A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci. Reports. Jan. 2020; 10(1): 1013–1039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamel NS, Gammack JK: Insomnia in the Elderly: Cause, Approach, and Treatment. Am. J. Med. Jun. 2006; 119(6): 463–469. Publisher Full Text\n\nMorin CM, et al.: Insomnia disorder. Nat. Rev. Dis. Primers. Sep. 2015; 1(1): 1–18. Publisher Full Text\n\nZhao Y, Singh C, Prober DA, et al.: Morphological and physiological interactions between GnRH3 and hypocretin/orexin neuronal systems in zebrafish (Danio rerio). Endocrinology. 2016; 157(10): 4012–4020. Publisher Full Text\n\nImperatore R, et al.: Overlapping distribution of orexin and endocannabinoid receptors and their functional interaction in the brain of adult zebrafish. Front. Neuroanat. 2018; 12(July): 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThangam EB, et al.: The Role of Histamine and Histamine Receptors in Mast Cell-Mediated Allergy and Inflammation: The Hunt for New Therapeutic Targets. Front. Immunol. Aug. 2018; 9(AUG). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar CP: Further Investigations on the Neuroprotective Potential of Centella siatica against Sleep Deprivation Induced Anxiety like Behaviour: Possible Implications of Mitoprotective and Anti-Stress Pathways. J. Sleep Disord. Treat. Care. 2017; 06(02). Publisher Full Text\n\nRoy D, Barman SK, Shaik M: Current Updates on Centella asiatica: Phytochemistry, Pharmacology and Traditional Uses. Med. Plant Res. 2013. Publisher Full Text\n\nProber DA, Rihel J, Onah AA, et al.: Hypocretin/Orexin Overexpression Induces An Insomnia-Like Phenotype in Zebrafish. J. Neurosci. Dec. 2006; 26(51): 13400–13410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYokogawa T, et al.: Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants. PLoS Biol. Oct. 2007; 5(10): e277–e2397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfif Z, Santoso MIE, Khotimah H, et al.: “Data supporting research results Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38. Effect of Centella asiatica ethanol extract on zebrafish larvae (Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38. 2023. (accessed Sep. 04, 2023). Reference Source\n\nAfif Z, Santoso MIE, Khotimah H, et al.: The ARRIVE guidelines 2.0: author checklist. Eff. Centella Asiat. ethanol Extr. zebrafish larvae (Danio rerio) insomnia Model through Inhib. Orexin, ERK, Akt p38. 2023. Publisher Full Text" }	[ { "id": "254619", "date": "27 Mar 2024", "name": "Desak Ketut Indrasari Utami", "expertise": [ "Reviewer Expertise Sleep disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFull report:\nComments for abstract: The research objective should also be stated in the abstract, so that it relates to the research results.\nIntroduction: The points made in the introduction are appropriate.\nMethod: The method is clear and has been approved by the ethical commission, but the reviewer does not understand the term media used, where in the manuscript it is stated that Centella asiatica extract is given once in 24 hours at the same time as the media is replaced. It is necessary to explain a little about this media.\nResults: It is clear. No special comments.\nDiscussion: The first paragraph of the discussion is not really necessary to discuss this research, so the discussion is more focused on the research results that have been obtained according to the research objectives to prove the hypothesis.\nComment on the answer to the question no. 1\nThe work is presented clearly and accurately, but some of the citations for this work are more than 5 years, there is even literature from year 2010.\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": "11340", "date": "13 Apr 2024", "name": "Annisatul Hakimah", "role": "Author Response", "response": "Comments for abstract: The research objective should also be stated in the abstract, so that it relates to the research results. Answer comments on the abstract section: We agreed to add a goal sentence to the abstract section \"The purpose of this study was to prove the effect of gotu kola extract (Centella asiatica) in improving the sleep activity of zebrafish larvae (Danio rerio) insomnia model, extending total inactivity time (cumulative duration) and shortening the duration of first sleep (latency to first) in light and dark phases through inhibition of orexin, ERK, p-38 and Akt\". Method: The method is clear and has been approved by the ethical commission, but the reviewer does not understand the term media used, where in the manuscript it is stated that Centella asiatica extract is given once in 24 hours at the same time as the media is replaced. It is necessary to explain a little about this media. Answer comments in the method section: We agree to add an explanation related to the media referred to in this study, namely the solution used as a medium for maintaining zebrafish embryos. The chemicals needed in the manufacture of medium embryonic stock solution prepared in 1 liter contained 10 grams of NaCl, 0.30 grams of KCl, 1,630 grams of MgSO4, 0.4 grams of CaCl. Discussion: The first paragraph of the discussion is not really necessary to discuss this research, so the discussion is more focused on the research results that have been obtained according to the research objectives to prove the hypothesis. Answering comments in the discussion section: In our opinion, in the initial paragraph of the discussion section, it needs to be explained because it is related to the process of observing the locomotor motion of zebrafish used in this study, namely the duration of video taking for 30 minutes of the light phase (end) and 30 minutes of the dark phase (beginning), the process of changing time from wakefulness to sleep. Comment on the answer to the question no. 1 The work is presented clearly and accurately, but some of the citations for this work are more than 5 years, there is even literature from year 2010. Answering the comment of question no.1: After we discussed the literature used in writing this article with several citations of works from the last 5 years because this study is the latest research on insomnia cases using zebrafish models, so for the latest literature we have not been able to find so far, but we can ensure that this work is presented clearly and accurately and by citing the latest literature in some related explanations." } ] }, { "id": "254621", "date": "24 Apr 2024", "name": "Naricha Chirakalwasan", "expertise": [ "Reviewer Expertise Sleep Medicine", "Pulmonary Medicine", "Critical Care 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\nAbstract - It is completed and summarized the rationale and the findings of the study. Introduction - It gave a good background on insomnia and underlying insomnia pathophysiology leading to this research project. Method-\nPlease clarify about how the authors calculated the number of embryos needed to be in the study. What was an alpha error and power that used for the calculation? The study result was demonstrated as mean +/- SD, please confirm that the data has normal distribution and test of normality was conducted.\nResult-\nI did not see the data on the number of zebrafish in each group, please give this information in the text and tables, were they 144 in each group? The meaning of superscript a,b, c should be clarified in the text and in each table so the readers understand what it is referring to and what group are being compared and whether the difference was statistically significant or not. In Table 5-8, the cumulative duration (day-second (s)), should be explained since the data was shown as xxx,xxxx. In table 5 and table 6, please explain why insomnia group had higher inactivity time (presumably sleep?) compared to normal group. In table 6, giving the dose of 5 microgram/ml CA resulting in increase in inactivity time, however the dose of 2.5 and 10 microgram/ml CA resulting in reduction in inactivity time, please explain since if the CA is really working, it should result in dose dependent effect (increasing the dose associated with improving or increasing inactivity time). In table 7 and 8, 5 microgram/ml CA resulting in latency to first light phase of 0 in all three days, however, some of the doses of 2.5 and 10 microgram/ml CA resulting in increase in latency to first light phase, please explain.\nDiscussion\nFirst paragraphs are not relevant and suggested to be removed. In the last paragraph (before the conclusion), “From this study, the average sleep latency at light ranged from 24.8 to 59.2 minutes on the 6th and 7th day experiments with light exposure during the day” this information does not match the data in table 7, please clarity.\nConclusion - It is concise.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "11508", "date": "18 Jul 2024", "name": "Annisatul Hakimah", "role": "Author Response", "response": "Abstract - It is completed and summarized the rationale and the findings of the study. Introduction - It gave a good background on insomnia and underlying insomnia pathophysiology leading to this research project. Method- 1. Please clarify about how the authors calculated the number of embryos needed to be in the study. What was an alpha error and power that used for the calculation? Response: The number of samples (n) in each treatment (p) is calculated based on the following formula (Solimun, 2001) with p = 7: p(n-1) ≥15 pn-p ≥ 15 7n-7 ≥ 15 7n ≥ 22 n ≥ 4 From the calculations obtained n ≥ 4, to anticipate the possibility of illness and death in fish, the number of samples (n) used was 6 fish larvae in each group. After optimizing the sample in the protein isolation process before WB testing, it was found that the sample needed for each group was n = 144 zebrafish larvae, in this study there were 5 treatment groups, so that the total sample needed was 720 zebrafish larvae. This is because the type of sample in the form of larvae is very small (7 dpf) so it requires a large enough number of samples for protein isolation. However, as many as 6 larvae were recorded to be examined for locomotor motion in observing the cycle of wake and sleep larvae. 2. The study result was demonstrated as mean +/- SD, please confirm that the data has normal distribution and test of normality was conducted. Response: All research data have been carried out statistical analysis of normality tests first before other tests and further tests. The data from the statistical analysis test has been listed in the Data availability point with DOI: 10.6084/m9.figshare.24078519. Data is available under the terms of the Creative Commons Attribution 4.0 International (CC-BY 4.0) license. Result- 1. I did not see the data on the number of zebrafish in each group, please give this information in the text and tables, were they 144 in each group? Response: The sample needed for each group is n = 144 zebrafish larvae, this study there are 5 treatment groups, so the total sample needed is 720 zebrafish larvae. For tables that have been corrected with an additional number of samples will be emailed separately. 2. The meaning of superscript a,b,c should be clarified in the text and in each table so the readers understand what it is referring to and what group are being compared and whether the difference was statistically significant or not. Response: Statistical analysis also showed that there was a significant difference in the value of orexin protein expression, this was evidenced by a sign of significance in the form of (a, b, c) which showed that exposure treatment to CA extract was proven to reduce the value of orexin protein expression significantly lower than normal and insomnia groups. The notations a, b, c indicate that there are significant differences between groups. The group with the notation a differs significantly from the group with the notation b, as well as the group with the nitation b differs significantly from the notation c, and vice versa the group with the notation a is also significantly different from the group with the notation c, and so on. 3. In Table 5-8, the cumulative duration (day-second (s)), should be explained since the data was shown as xxx,xxxx. Response: We will fix this in table sections 5-8. However, in tables 5-8 on the journal website, cumulative duration data figures have been displayed. Please inform us about this. 4. In table 5 and table 6, please explain why insomnia group had higher inactivity time (presumably sleep?) compared to normal group. Response: Light has a very strong effect on suppressing sleep in zebrafish (Yokogawa et al., 2007). By the time zebrafish are given light exposure, zebrafish will not sleep at all. Light can suppress the sleep of zebrafish. Zebrafish are sensitive to light and light suppresses the production of melatonin, a hormone that stimulates sleep in zebrafish. Light strongly suppresses zebrafish sleep by up to 90% causing a decrease in sleep. The rate of sleep disturbance may be good but sleep disturbance with light may still cause nonspecific effects through sensory stimulation and changes in the circadian clock (Bringmann, 2019). Our results showed that on day 5 zebrafish larvae still need circadian rhythm adjustment compared to days 6 and 7. 5. In table 6, giving the dose of 5 microgram/ml CA resulting in increase in inactivity time, however the dose of 2.5 and 10 microgram/ml CA resulting in reduction in inactivity time, please explain since if the CA is really working, it should result in dose dependent effect (increasing the dose associated with improving or increasing inactivity time). Response: Based on the results of this study, it can be concluded that the increase or decrease that occurs in the measured parameters in this study is related to the concentration of Centella asiatica extract, classified as still safe for oral consumption. From this study it can be seen that the most optimal concentration in resulting in increased inactivity time is at a concentration of 5 micrograms / ml CA. However, further research is needed to ensure the safety of concentration and the most effective dose of Centella asiatica extract, especially in insomnia conditions and several protein molecules that play a role in sleep activity. Centella asiatica has a role as a therapy for sleep disorders, one of which is by inhibiting histamine. Histamine itself is a neurotransmitter in the brain that plays a role in waking up. Histamine binding to histamine H3 receptors activates Gαi/o signaling causing MAPK activation. H3R activation is involved in sleep-wake cycles, recognition, regulation of homeostasis of energy levels, and neurotransmission (Thangam et al., 2018). 6. In table 7 and 8, 5 microgram/ml CA resulting in latency to first light phase of 0 in all three days, however, some of the doses of 2.5 and 10 microgram/ml CA resulting in increase in latency to first light phase, please explain. Response: Based on the results of this study, it can be concluded that the increase or decrease that occurs in the measured parameters in this study is related to the concentration of Centella asiatica extract, classified as still safe for oral consumption. However, further research is needed to ensure the safety of concentration and the most effective dose of Centella asiatica extract, especially in insomnia conditions and several protein molecules that play a role in sleep activity. Discussion- 1. First paragraphs are not relevant and suggested to be removed. Response: We appreciate the reviewer's suggestion to remove the first paragraph in the discussion section, but our purpose in that paragraph is to provide an explanation regarding references to locomotor motion observation methods related to zebrafish wake and sleep cycles. 2. In the last paragraph (before the conclusion), “From this study, the average sleep latency at light ranged from 24.8 to 59.2 minutes on the 6th and 7th day experiments with light exposure during the day” this information does not match the data in table 7, please clarity. Response: The explanation in this paragraph leads to reference literature of other journals that we refer to as support for the results of our research. Conclusion - It is concise. Reference- E. B. Thangam et al., “The Role of Histamine and Histamine Receptors in Mast Cell-Mediated Allergy and Inflammation: The Hunt for New Therapeutic Targets,” Front. Immunol., vol. 9, no. AUG, Aug. 2018, doi: 10.3389/FIMMU.2018.01873. H. Bringmann, “Genetic sleep deprivation: using sleep mutants to study sleep functions,” EMBO Rep., vol. 20, no. 3, pp. 1–14, 2019, doi: 10.15252/embr.201846807. T. Yokogawa et al., “Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants,” PLoS Biol., vol. 5, no. 10, pp. 2379–2397, Oct. 2007, doi: 10.1371/JOURNAL.PBIO.0050277." } ] }, { "id": "254620", "date": "28 May 2024", "name": "Rimawati Tedjasukmana", "expertise": [ "Reviewer Expertise Sleep disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is scientifically sound, with good methods and valid results. However, parts of it are lost in translation because of the writing. A. Some paragraphs are confusing. For example: 1. Paragraph 3 of Introduction is a very important paragraph, because it explained the reasons behind the authors' decision to choose the proteins tthat they investigated. This paragraph is difficult to follow, therefore it needs a complete rewrite and better still adding a diagram will be very enlightening. 2. The whole discussion needs a rewrite, because the authors went off in a tangent in this part. It is better to stick to a few important points, i.e state the findings of the study and elaborate, compare the findings with results of other studies, analyse the discrepancies.\nB. Confusing use of phrases. For example 1. Latency to first (page 10 paragraph 2). It is better suited to use the phrase 'latency to first sleep' 2. Total time of first sleep (page 10 paragraph 2). The authors were trying to explain 'latency to first'. It is better to use 'time to first sleep onset'.\nC. Some uncommonly used abbreviations have no explanations. and sometimes the authors forgot to write the units after some numbers in the results part\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": "11709", "date": "18 Jul 2024", "name": "Annisatul Hakimah", "role": "Author Response", "response": "This article is scientifically sound, with good methods and valid results. However, parts of it are lost in translation because of the writing. A. Some paragraphs are confusing. For example: 1. Paragraph 3 of Introduction is a very important paragraph, because it explained the reasons behind the authors' decision to choose the proteins tthat they investigated. This paragraph is difficult to follow, therefore it needs a complete rewrite and better still adding a diagram will be very enlightening. Answer: we will list images related to the signal path of the Orexin system. We will send you pictures via email. 2. The whole discussion needs a rewrite, because the authors went off in a tangent in this part. It is better to stick to a few important points, i.e state the findings of the study and elaborate, compare the findings with results of other studies, analyse the discrepancies. Answer: We have explained in the section related to important research findings that such as proteins used as markers, have proven their role in the mechanism of insomnia disease or circadian rhythm cycles (wake and sleep). And we have also compared the findings in this study which were later corroborated with other previous research theories to confirm the results of our study. B. Confusing use of phrases. For example 1. Latency to first (page 10 paragraph 2). It is better suited to use the phrase 'latency to first sleep' Answer: we will change the phrase \"Latency to first\" to \"latency to first sleep\". 2. Total time of first sleep (page 10 paragraph 2). The authors were trying to explain 'latency to first'. It is better to use 'time to first sleep onset'. Answer: we will change the phrase \"Total time of first sleep\" to \"time to first sleep onset\". C. Some uncommonly used abbreviations have no explanations. and sometimes the authors forgot to write the units after some numbers in the results part Answer: because we have included units in the table and diagram of results, so we do not include in the explanation of results section, if it is necessary to include units in the explanation of results section, we will correct it." } ] } ]	1	https://f1000research.com/articles/13-107
https://f1000research.com/articles/11-1272/v1	08 Nov 22	{ "type": "Research Article", "title": "Comparative analysis of computed tomography severity indices in predicting the severity and clinical outcome in patients with acute pancreatitis", "authors": [ "Geetanjali Parmar", "Griselda Philomena Noronha", "Vinaya Poornima", "Geetanjali Parmar", "Vinaya Poornima" ], "abstract": "Background: Acute pancreatitis (AP) has unpredictable severity. Its management is based on initial assessment of disease severity. It ranges from mild interstitial to severe necrotic form; the latter is associated with poor prognosis. Contrast-enhanced computed tomography (CT) of the abdomen is the gold standard in early detection of pancreatic necrosis and in assessing the severity of AP. Two CT grading systems exist to assess severity of AP: CT severity Index (CSI) and modified CSI (MCSI). This study compares the usefulness of these two systems in predicting severity and clinical outcome in AP in comparison with Ranson’s criteria and clinical outcome parameters. Methods: This is a prospective hospital-based screening study of 80 patients aged >12 years with clinical diagnosis of AP who underwent contrast-enhanced CT study of the abdomen. Comparative analysis between MCSI and CSI with Ranson’s criteria and clinical outcome parameters was assessed by Chi-Squared test. Results: The accuracy of CSI and MSCI in predicting the requirement of critical care, superadded infection, multiple organ dysfunction syndrome (MODS) and requirement of intervention were 73.0%, 64.5%, 69.8% 60.9% and 77.2%, 76.0%, 74.4% & 56.6%, respectively. Area under the curve for MCSI score was significantly higher (AUC : 0.861; 95% CI: 0.736-0.986) than CSI score (AUC:0.815;95% CI:0.749-0.941). MCSI and CSI showed significant correlation with Ranson’s criteria; however, MCSI correlation was better (r:0.53; p<0.01) than CSI (r:0.35;p:0.04).\nConclusion: CSI and MCSI are better predictors of severity, clinical outcome and mortality compared with Ranson’s criteria, with MCSI being more accurate and better predictor than CSI. The accuracy of MCSI is better than CSI for prediction of requirement of critical care, development of superadded infection and development of MODS in AP. However, CSI and MCSI have low accuracy in predicting intervention in AP.", "keywords": [ "Acute Pancreatitis", "CT severity index", "Modified CT severity index", "clinical outcome parameters", "Ranson’s criteria", "hospital stay", "multisystem organ dysfunction syndrome", "sepsis." ], "content": "Introduction\n\nAcute pancreatitis (AP) is one of the most common causes of acute abdomen with unpredictable clinical course. Based on the severity, 80% of cases are mild and 20% of cases are severe which morphologically correlate with edematous and necrotizing forms of AP respectively. The mild form is self-limiting without causing major physiological insult. The severe form is life threatening and can lead to early or late multiple organ dysfunction syndrome (MODS) and superadded infection.1–3\n\nContrast enhanced computed tomography (CT) of the abdomen is the gold standard4 in identifying necrosis and fluid collections in AP which can aid in predicting disease severity and prognosis of the patient, thus guiding the management. Various studies suggest the evidence that severity can be better assessed by CT than the numerical grading systems due to direct visualization of necrosis and complications of AP on CT.5,6 Although the CT severity index (CSI) shows good correlation with the severity of AP, few studies suggested few limitations. CSI doesn’t show good correlation with clinical outcome, mortality, need for surgical or percutaneous interventional procedures, MODS and superadded infection.3 These shortcomings led to the modification and simplification of CSI by Mortele et al.3 leading to the formation of modified CT severity index (MCSI). The present study is comparative analysis of MCSI and CSI with clinical outcome parameters and Ranson’s criteria in patients with AP.\n\n\nMethods\n\nThis is a prospective hospital-based screening study performed in the department of Radiodiagnosis affiliated to Kasturba Medical College (KMC), Mangalore (MLR), Manipal Academy of Higher Education (MAHE), Manipal, India on 80 patients with clinical diagnosis of AP who underwent contrast enhanced CT abdomen over a period of 2 years from September 2019 to September 2021. The study was performed after the approval from the Institutional Ethics Committee (IEC), KMC, MLR, MAHE with approval number of IEC KMC MLR 09-19/411.\n\nPaediatric patients <12 years of age were excluded from the study, as Ranson’s criteria is not done in this group of patients in our hospital. Patients with poor imaging results due to poor compliance or motion artefacts were excluded. Patients without intravenous (i.v.) contrast administration were also excluded. Patients with diagnosis of acute-on-chronic, recurrent and calcific pancreatitis and those that got discharged against medical advice or were lost to follow up were also excluded from the study. Patients with cardiac, renal & respiratory comorbidities were excluded from the study. Since the informed consent is routinely taken prior to every CT study and research data are obtained from the CT machine computer and patient case files with no direct interaction with the study participants, IEC, KMC, MLR waived off additional informed consent from the study participants for this research.\n\n16-slice and 32-slice CT scanner machines were used to acquire 5-mm plain CT axial sections followed by the administration of 1.5–2.0 mL/kg body weight (80–100 mL) of non-ionic i.v. contrast through the automated injector. This was followed by around 1 mL/kg body weight (40–50 mL) of normal saline. The rate of injection for both contrast and saline administration was ~4 mL/s which was altered in accordance with haemodynamic status, body weight and size of the i.v. cannula. The images were acquired in the arterial and porto-venous phases at 6–8 and 35–45 seconds respectively in all cases by bolus tracking method which is described as follows. A locator was placed on the aorta at D12–L1 level and the contrast injection got automatically triggered via the automated injector once the aorta at this level showed optimum contrast opacification. Axial sections of 5 mm slice thickness were then reformatted to thin 0.6 mm axial, sagittal and coronal sections. The clinical and laboratory details of the patient were obtained from the CT requisition form and patient case file. This was followed by assessment of severity of acute pancreatitis using both CSI (Tables 1a, 1b and 1c) and MCSI (Tables 2a, 2b and 2c). Accordingly, severity of AP was graded as mild, moderate and severe based on the scores.\n\nWherever available Ranson’s criteria score was noted down from patient case file and the correspondence of both the CT indices were studied with respect to the Ranson’s criteria. Ranson’s criteria score consists of 11 prognostic parameters, out of which five parameters are assessed at the admission and six parameters are assessed during initial 48 hours of hospital stay (Tables 3a and 3b).\n\nRanson’s score of 0 or 1 suggests complications will not develop in AP and mortality is negligible. On the other hand, Ranson’s score of 3 or more predicts severe AP with possible mortality.9 The mortality in AP is directly proportional to the Ranson’s criteria score (Table 3c).\n\nThe clinical outcome parameters6,7 were noted down from all the patient case files and its association with CT severity indices were studied and are as follows:\n\n1. The extent of hospital or intensive care unit (ICU) stay (greater than or equal to 15 days);\n\n2. Requirement of critical care, (Arterial oxygen tension (PO2) <60 mmHg or requirement of ventilation, systolic blood pressure (BP) <90 mmHg);\n\n3. Requirement for (surgical/percutaneous) intervention (like drainage and aspiration);\n\n4. Evidence of infection, (combination of a fever more than 100°F and elevated WBC count greater than 15,000 cells/mm);\n\n5. Existence of organ failure (Arterial PO2 <60 mmHg or requirement of ventilation, serum creatinine of >3 mg/dL or urine output of <500 mL per 24 h and systolic BP of <90 mmHg); and\n\n6. Death.\n\nOutcome Variables that were studied are as follows:\n\n• Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of CSI and MCSI with respect to clinical outcome parameters like mean hospital stay, requirement of critical care, superadded infection, MODS, requirement of intervention & mortality.\n\n• Concordance of CSI and MCSI with the score of Ranson’s criteria.\n\nThe data was collected on a pre-designed study proforma. Qualitative data was expressed as percentage and frequency. Chi-Squared test was used to assess the association among the qualitative variables. The level of significance was represented by p-value of less than 0.05. Screening efficacy was computed using standard formulae. Wherever necessary, the results were graphically represented. Pearson correlation was used to assess the magnitude and direction of association between CSI and MCSI with Ranson’s score. Receiver operating characteristics (ROC) curves were used to compare the role of CSI and MCSI in predicting the mortality in AP with r value from +1 to −1. The r value of +0.1 to +1, 0 and −0.1 to −1 was suggestive of positive, zero and negative correlation respectively. Area under the curve (AUC) between CSI and MCSI as predictor of mortality was analyzed. Statistical package for social sciences (SPSS) version 21.0 (RRID:SCR_002865) and Microsoft Excel 2010 (RRID:SCR_016137) were used for most of the analysis and graphical representation respectively.\n\n\nResults\n\nThe patients with AP in this study were more or less equally distributed across all the decades from 2nd to 6th decade with mean age of 44.41 years. There was clear male predominance of 77.5% with 22.5% female patients with male to female ratio of 3.5:1. The most common cause for acute pancreatitis was alcoholism (56.3%) followed by gall stones (28.8%).\n\nAs per MCSI, more than half of patients (56%) with AP had mild disease, about one third of them (36.3%) had moderate disease and a small percentage (7.5%) had severe disease (Figure 1a).\n\nAs per CSI, about half of patients (52.5%) with AP had moderate disease, about one fourth of them (26.3%) had mild disease and 21.3% had severe disease (Figure 1b).\n\nRequirement of critical care\n\nBased on MCSI score, all the patients with severe AP (100.0%) required critical care, 82.8% of moderate disease needed critical care and only one third of patients with mild disease (31.1%) needed intensive care with (p<0.01) (Figure 2a). The overall sensitivity and specificity for prediction of requirement of critical care was 85.7% and 68.9% respectively with an accuracy of 77.2%.\n\nAs per CSI score, most (95.2%) of moderate disease, about half (52.4%) of severe disease and small percentage (11.8%) of mild disease required critical care (p<0.01) (Figure 2b). The overall sensitivity and specificity for prediction of critical care requirement was 66.7% and 88.2% respectively with an accuracy of 73%.\n\nAs per MCSI, the superadded infection was seen in 83%, 41% and 4% of severe, moderate and mild disease of AP respectively (p value<0.01) (Figure 3a). The overall sensitivity and specificity were 49% & 96% respectively with an accuracy of 76% in predicting superadded infection in AP patients.\n\nBased on the CSI score, there was no (0.0%) superadded infection in mild disease, while it was present in slightly less than half (47.6%) of severe disease and about 21.4% in moderate disease (p<0.01) (Figure 3b). Hence the overall specificity & sensitivity for prediction of presence of superadded infections was 100.0% and 30.2% respectively with accuracy of 64.5%.\n\nAs per MCSI, MODS developed in 15.6% of mild, 58.6% of moderate and 83.3% of severe AP (p<0.01) (Figure 4a). Overall sensitivity & specificity for prediction of development of MODS were 62.9% and 84.4% respectively with an accuracy of 74.4% respectively.\n\nAs per the CSI score, there was no (0.0%) development of MODS in mild disease. On the contrary, most (95.2%) of severe disease and 21.4% percentage of moderate disease developed MODS (Figure 4b). The overall specificity & sensitivity for prediction of development of MODS was 100.0% and 40.0% respectively with an accuracy of ~69.8%.\n\nAs per MCSI, intervention was performed in 55.6% of mild, 65.5% of moderate & 83.3% of severe cases of AP (p<0.01) (Figure 5a). The overall sensitivity & specificity for prediction of requirement of intervention was 68.6% and 44.4% respectively with an accuracy of 56.6%.\n\nAs per CSI score, approximately three-quarters (76.2%) of patients with severe acute pancreatitis required intervention, 61.9% of patients with moderate disease and 41.2% of patients with mild disease required intervention (p<0.01) (Figure 5b). The overall sensitivity and specificity for prediction of requirement of intervention by CSI was ~66.7% and ~58.8%, respectively, with an accuracy of ~60.9%.\n\nMCSI score showed both good sensitivity and specificity for development of MODS, good sensitivity for prediction of requirement of critical care & intervention. MCSI showed good specificity for the development of superadded infection (Figure 6a).\n\nCSI score showed high specificity for the development of MODS and superadded infection. The overall accuracy is better with MCSI score than CSI score for prediction of requirement of critical care, development of superadded infection & development of MODS. Both MCSI and CSI scores had low accuracy in predicting the requirement of intervention (figure 6b).\n\nAs per MCSI score, mortality rate was 100.0% in AP, 17.2% in moderate disease and 2.2% in mild disease (Figure 7a). The overall sensitivity and specificity for prediction of mortality was 91.7% and 64.7%, respectively, with an accuracy of 87.5%.\n\nAs per CSI score, mortality rate was one-third (33.3%) and highest in severe grade of AP, followed by mild grade of AP (17.6%) with lowest mortality rate in moderate grade (4.8%) (Figure 7b). The overall sensitivity & specificity for prediction of mortality was 58.5% and 79.4%, respectively, with an accuracy of 73.8%.\n\nThe mean hospital stay by MCSI was highest in moderate grade of AP with ~22 days as compared to approximately 12 & 13 days in mild & severe disease respectively (p<0.01) (Table 4a).\n\nThe mean hospital stay as per CSI score was significantly higher in moderate and severe grade of acute pancreatitis corresponding to approximately 18 and 19 days respectively as opposed to approximately 8 days in mild disease (p<0.01) (Table 4b).\n\nRanson’s criteria score was available with 31 out of 80 patients (38.8%). There was significant correlation between Ranson’s criteria and both CT severity indices (CSI and MCSI) but the correlation was highly statistically significant and better with MCSI score (r=0.53; p<0.01) as compared to CSI score (r=0.35; p=0.04) (Table 5, Figures 8a & 8b).\n\nAccording to ROC Curve analysis, both CSI & MCSI scores were significant predictors of development of mortality in AP. However, area under curve was significantly higher for MCSI score (AUC 0.861; 95% CI 0.736–0.986) as compared to CSI score (AUC 0.815; 95% CI 0.749–0.941) (Table 6 & Figure 9).\n\n\n\n\nDiscussion\n\nContrast enhanced computed tomography of the abdomen is the imaging modality of choice and is the gold standard in the diagnosis of AP. The necrotizing form of AP, though less common, if present, is associated with a myriad of life-threatening complications. Among all the diagnostic tests available, CT has the highest diagnostic accuracy in detecting pancreatic necrosis.11\n\nSteinberg et al.12 in their study suggested the evidence of 80 to 90% of AP was due to cholelithiasis & chronic alcoholism. Our study suggests evidence of alcoholism as the most common etiological factor for AP (56.3%) followed by gall stones (28.8%). Similar evidence was suggested by Wongnai et al.13 in their study on 90 patients of AP, where alcoholism and pancreatico-biliary ductal calculi were reported as aetiological factor in 60% and 18% patients respectively. In India, alcohol consumption is predominantly seen in males (male to female ratio of 24.3:1).14 The suggestive evidence of alcohol abuse as the commonest aetiological factor of AP combined with the male predominance of alcohol consumption in India explains the male to female preponderance (3.5:1) in this study. Similar evidence was suggested by Dugernier T L et al.15 and Balthazar EJ et al.16\n\nOn the contrary, Raghuwanshi S et al.17 suggested the evidence of most common aetiology for AP as cholecystolithiasis (42%) followed by alcoholism (38%) with remaining 20% aetiology for AP belonged to rest category which included idiopathic, trauma and drug induced cases (24%, 2% and 2% respectively). Casas et al.18 in their study on 148 patients suggested cholelithiasis (57%) as the most common aetiological factor for AP followed by alcoholism (21%) with both together contributing to another 5% of AP patients. Bollen TL et al.19 and Jauregui et al.20 also suggested the evidence of cholelithiasis as the predominant aetiological factor for AP.\n\nThis study is comparative analysis between MCSI and CSI grading systems in assessing severity and clinical outcome. Majority of the patients with AP belonged to mild category as per MCSI and moderate category as per CSI. This resulted in a small group of patients who had different category of severity by CSI and MCSI. The present study suggests MCSI to be more accurate predictor of severity than CSI as it predicted clinical outcome more accurately in those patients who were differently categorized in severity by CSI. This better prediction of severity and clinical outcome by MCSI in AP may be attributable to inclusion of extra-pancreatic complications of AP like ascites, pleural effusion, vascular complications and gastrointestinal complications in the assessment of MCSI which are not included in CSI. Kondekar S et al.7 and Banday et al.21 suggested partially opposing evidence from our study where majority of the patients by MCTSI belonged to mild category as per our study and the majority of the patients belonged to severe category as per CSI unlike our study.\n\nBanday et al.21 in their study suggested evidence of increasing mean duration of hospital stay with increasing severity by MCTSI score and concluded that the duration of mean hospital stay is directly proportional to severity grading by MCTSI system in acute pancreatitis.\n\nOur study suggests mean hospital stay in AP by CSI score is significantly longer in moderate and severe disease as compared to mild disease (p<0.01) whereas the mean hospital stay by MCSI is significantly longer in moderate disease as compared to mild and severe disease (p<0.01). This can be attributed to the fact that mild cases were discharged relatively early from hospital in comparison to moderate category cases and very severe cases had higher mortality with lesser hospital stay.\n\nOverall in the present study, MCSI score showed good sensitivity for prediction of requirement of critical care, development of MODS and requirement of intervention. MCSI showed good specificity for MODS and development of superadded infection. CSI showed high specificity for MODS and development of superadded infection. Overall accuracy of MCSI was better than CSI for prediction of requirement of critical care, development of superadded infection and development of MODS. Both scores showed lower accuracy with regard to requirement of intervention.\n\nThe sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MCSI in predicting severity according to the study by Bollen TL et al.19 were 71%, 93%, 69% and 94%, respectively. This study suggested evidence of accurate correlation of clinical scoring systems with systemic complications & mortality in acute pancreatitis. The study also suggested evidence that radiological scoring system was more accurate in predicting the severity of acute pancreatitis, superadded infection and need for intervention than clinical scoring system. Among the two radiological scoring systems, the study suggested no evidence of significant differences between CSI and MCSI in predicting severity in acute pancreatitis.\n\nBollen et al.22 suggested CSI showed better sensitivity, specificity, PPV and NPV than MCSI. Whereas Jauregui-Arrieta LK et al.19 suggested different evidence where MCSI showed better sensitivity, specificity and PPV than CSI in severe AP and concluded that MCSI is better screening test than CSI in severe AP. Sharma et al.23 performed suggested sensitivity and NPV is better with MCSI (98.6% and 90%, respectively) than CSI (87.3% and 57.1%, respectively) with similar PPV for both (~74%) and low specificity of 26.5% and 35.3% for MCSI and CSI, respectively.\n\nThe present study shows significant correlation between Ranson’s criteria and both severity indices on CT (CSI and MCSI) but the correlation of MCSI with Ransons’ criteria is highly statistically significant which suggests MCSI as a better predictor of severity and clinical outcome than CSI.\n\nOn receiver operating characteristic (ROC) curve analysis, the present study suggests evidence that both CSI and MCSI are significant predictors of development of mortality in AP. However, the area under curve was significantly higher for MCSI score (AUC 0.861; 95% CI 0.736–0.986) as compared to CSI (AUC 0.815; 95% CI 0.749–0.941) which suggests MCSI as better predictor of mortality in AP than CSI.\n\nMangalanandan S et al.24 suggested evidence of strong correlation between Ranson’s criteria and MCSI with mild and severe forms of AP showing 100% agreement with each other. But moderate category in MCSI Score had disagreeing results because Ranson’s criteria has only mild and severe categories due to which moderate category patients could not be studied. Their study suggested that MCSI (sensitivity of 93.33% and specificity of 54.17%) is more sensitive but less specific than Ranson’s criteria (sensitivity of 80% and specificity of 83.3%) in predicting actual outcome of AP. Although Chand P et al.25 suggested evidence of lack of statistical significant difference between Ranson’s criteria and MCSI in evaluation of the outcome of AP with respect to the systemic complications,7 there was statistically significant difference between MCSI and Ranson’s criteria with respect to local complications with increased incidence of local complications with higher Ranson’s criteria.\n\nOne of the drawbacks in the study was pediatric patients below the age of 12 years were not concluded as Ranson’s criteria is not done for them in our hospital. Also, larger sample size will reduce the margin of error in the study. Third drawback is the lack of availability of Ranson’s criteria score in 61.3% of the patients in the study due to usage of various other alternative clinical grading systems like Revised Atlanta Classification, Acute physiology and chronic health evaluation (APACHE) II & Bedside index of severity in acute pancreatitis (BISAP) by the treating clinician. These alternative clinical grading systems are affordable, quick & requires less effort in assessing the severity of AP than Ranson’s criteria.\n\n\nConclusion\n\nBoth CSI and MCSI are better predictors of severity, clinical outcome and mortality than Ranson’s criteria in patients with AP with MCSI being more accurate & better predictor of the same than CSI. The accuracy of MCSI is better than CSI for prediction of requirement of critical care, development of superadded infection and development of MODS. Both CSI and MCSI scores have low accuracy with regard to requirement of intervention in AP patients.\n\n\nData availability\n\nMendeley: Underlying data for ‘Comparative analysis of computed tomography severity indices in predicting the severity and clinical outcome in patients with acute pancreatitis’, https://doi.org/10.17632/htkkzr9zbr.2.26\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgment\n\nWe would like to acknowledge Dr. Ashvini Kumar, Former Head of Department & Former Professor of Department of Radiodiagnosis, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India who provided insight & expertise that greatly assisted this research.\n\n\nReferences\n\nFrossard JL, Steer ML, Pastor CM: Acute pancreatitis. Lancet. 2008; 371: 143–152. Publisher Full Text\n\nBalthazar EJ: Acute Pancreatitis: Assessment of Severity with Clinical and CT Evaluation. Radiology. June 2002; 223(3): 603–613. Publisher Full Text\n\nMortele KJ, Wiesner W, Intriere L, et al.: A Modified CT Severity Index for Evaluating Acute Pancreatitis: Improved Correlation with Patient Outcome. AJR. 2004; 183: 1261–1265. PubMed Abstract \| Publisher Full Text\n\nZhao K, Adam SZ, Keswani RN, et al.: Acute Pancreatitis: Revised Atlanta Classification and the Role of Cross-Sectional Imaging. AJR. 2015; 205: W32–W41. PubMed Abstract \| Publisher Full Text\n\nLeung TK, Lee CM, Lin SY, et al.: Balthazar computed tomography severity index is superior to Ranson criteria and APACHE II scoring system in predicting acute pancreatitis outcome. World J. Gastroenterol. 2005; 11(38): 6049–6052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar AH, Griwan MS: A comparison of APACHE II, BISAP, Ranson’s score and modified CTSI in predicting the severity of acute pancreatitis based on the 2012 revised Atlanta Classification. Gastroenterol. Rep (Oxf). 2018 May; 6(2): 127–131. PubMed Abstract \| Publisher Full Text\n\nKondekar S, Minne I: Assessment of acute pancreatitis using CT severity index and modified CT severity index: A tertiary care hospital based observational study. Int. J. Radiol. Diagn. Imaging. 2020; 3(1): 118–122. Publisher Full Text\n\nAbu-Eshy SA, Abolfotouh MA, Nawar E, et al.: Ranson's criteria for acute pancreatitis in high altitude: do they need to be modified? Saudi J. Gastroenterol. 2008 Jan; 14(1): 20–23. PubMed Abstract \| Publisher Full Text\n\nBasit H, Ruan GJ, Mukherjee S:Ranson Criteria. [Updated 2021 Sep 28]. StatPearls. Treasure Island (FL):StatPearls Publishing; 2022 Jan.Reference Source\n\nBhat MS: SRB’s manual of surgery. 6th ed.India:Jaypee Brothers Medical Publishers (P) Ltd.;2019.\n\nUrooj T, Shoukat S, Bokhar I, et al.: Diagnostic accuracy of contrast enhanced computed tomography (CECT) in detection of necrosis in acute pancreatitis by taking surgical findings as gold standard. J. Pak. Med. Assoc. November 2020; 70(11): 1930–1933.\n\nSteinberg WM, Chari ST, Forsmark CE, et al.: Controversies in clinical pancreatology: management of acute idiopathic recurrent pancreatitis. Pancreas. 2003 Aug; 27(2): 103–117. Publisher Full Text\n\nWongnai A, Mai WN: CT FINDINGS OF ACUTE PANCREATITIS IN MAHARAJ NAKORN CHIANG MAI HOSPITAL. Chiang Mai Med. J. 2007; 46(2): 45–53.\n\nBalasubramani K, Paulson W, Chellappan S, et al.: Sociodemographic Risk Factors of Alcohol Consumption in Indian Men and Women: Analysis of National Family Health Survey-4 (2015-16), a Nationally Representative Cross-Sectional Study Front. Public Health. August 2021; 9: 1–10. Publisher Full Text\n\nDugernier TL, Laterre PF, Wittebolr X, et al.: Compartmentalization of the inflammatory response during acute pancreatitis: correlation with local and systemic complications. Am. J. Respir. Crit. Care Med. 2003 Jul; 168(2): 148–157. PubMed Abstract \| Publisher Full Text\n\nBalthazar EJ, Freeny PC, VanSonnenberg E: Imaging and intervention in acute pancreatitis. Radiology. 1994 Nov; 193(2): 297–306. Publisher Full Text\n\nRaghuwanshi S, Gupta R, Vyas MM, et al.: CT Evaluation of Acute Pancreatitis and its Prognostic Correlation with CT Severity Index. J. Clin. Diagn. Res. 2016 Jun; 10(6): TC06–TC11. PubMed Abstract \| Publisher Full Text\n\nCasas JD, Díaz R, Valderas G, et al.: Prognostic value of CT in the early assessment of patients with acute pancreatitis. AJR Am. J. Roentgenol. 2004 Mar; 182(3): 569–574. PubMed Abstract \| Publisher Full Text\n\nBollen TL, Singh VK, Maurer R, et al.: Comparative evaluation of the modified CT severity index and CT severity index in assessing severity of acute pancreatitis. AJR Am. J. Roentgenol. 2011 Aug; 197(2): 386–392. PubMed Abstract \| Publisher Full Text\n\nJáuregui-Arrieta LK, Alvarez-López F, Cobián-Machuca H, et al.: Effectiveness of the modify tomographic severity index in patients with severe acute pancreatitis. Rev. Gastroenterol. Mex. 2008; 73(3): 144–148. PubMed Abstract\n\nBanday IA, Gattoo I, Khan AM, et al.: Modified Computed Tomography Severity Index for Evaluation of Acute Pancreatitis and its Correlation with Clinical Outcome: A Tertiary Care Hospital Based Observational Study. J. Clin. Diagn. Res. 2015 Aug; 9(8): TC01–TC05. PubMed Abstract \| Publisher Full Text\n\nBollen TL, Singh VK, Maurer R, et al.: A comparative evaluation of radiologic and clinical scoring systems in the early prediction of severity in acute pancreatitis. Am. J. Gastroenterol. April 2012; 107(4): 612–619. PubMed Abstract \| Publisher Full Text\n\nSharma V, Rana SS, Sharma RK, et al.: A study of radiological scoring system evaluating extrapancreatic inflammation with conventional radiological and clinical scores in predicting outcomes in acute pancreatitis. Ann. Gastroenterol. 2015; 28(3): 399–404. PubMed Abstract\n\nMangalanandan S, Thomas DA, Benjamin G: Correlation of Modified Computed Tomography Severity Index with Ranson’s Criteria in Assessing Severity of Acute Pancreatitis. Int. J. Anat. Radiol. Surg. 2021 Jan; 10(1): RO22–RO27. Publisher Full Text\n\nChand P, Singh R, Singh DP, et al.: Evaluation of the Outcome of Acute Pancreatitis by Ranson’s Criteria and Modified CT Severity Index. International Journal of Contemporary Medicine Surgery and Radiology. 2017; 2(2): 58–61.\n\nNoronha G, Parmar G, Poornima V: “COMPARITIVE ANALYSIS OF CT SEVERITY INDICES IN PREDICTING THE SEVERITY & CLINICAL OUTCOME IN PATIENTS WITH ACUTE PANCREATITIS”, Mendeley [DATASET].2022; V2. Publisher Full Text" }	[ { "id": "182028", "date": "19 Jul 2023", "name": "Saurabh Dawra", "expertise": [ "Reviewer Expertise Pancreas", "Liver" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nI would recommend to change the first line that the disease has unpredicted severity. With the present level of research, we do have fair enough idea of predicted severity. The authors may rephrase the sentence.\n\nWhile it is agreed that CTSI has it’s fair share of limitations, the same hasn’t been brought about clearly in the “Introduction“ section.\n\nPlease bring out clearly why have you chosen Hanson’s criteria as comparison to CTSI. What about Atlanta classification?\n\nMethods:\nHow was pancreatitis diagnosed?\n\nWhat is your institutional protocol of managing pancreatitis?\n\nAt what stage of disease/ day of illness was the CT done?\n\nDo you perform all investigations as required to calculate Ranson’s criteria on all patients with AP?\n\nWhat do you mean by critical care? What is your institute’s protocol in admitting the patients for critical care?\n\nDiscussion:\nWhat is unique in your study? How is it different from other comparisons including meta analysis on the same topic?\n\nWhat are your 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11286", "date": "04 Apr 2024", "name": "Griselda Noronha", "role": "Author Response", "response": "Thank you for your insightful questions regarding our manuscript. We appreciate the opportunity to clarify these points and ensure a comprehensive understanding of our study protocols and findings. Below, please find detailed responses to each of your queries: introduction: \"I would recommend to change the first line that the disease has unpredicted severity. With the present level of research, we do have fair enough idea of predicted severity. The authors may rephrase the sentence\"- Thank you so much for bringing out this to us. we will surely rephrase this sentence. \"While it is agreed that CTSI has it’s fair share of limitations, the same hasn’t been brought about clearly in the “Introduction“ section\" - Again thank you for your valuable feedback. we shall do this change. \"Please bring out clearly why have you chosen Hanson’s criteria as comparison to CTSI. What about Atlanta classification?\" a)The Ranson criteria was the first scoring system specifically designed to assess the severity and prognosis of acute pancreatitis & hence it is extensively studied and validated globally. Since its used since ages, it is a reliable tool. b) Ranson’s score is directly correlated with patient outcomes which means it is a clear prognostic indicator. High score at admission and at 48hours later is associated with increased rate of mortality and morbidity which helps in stratifying patients according to the need for ICU admission and aggressive therapy. c) Ranson criteria is simpler and more straightforward, making it easier to apply in clinical settings, especially where immediate decisions are required while other scores like APACHE II requires more amount of data & is relatively complex to calculate. d) APACHE II and BISAP are non-specific to pancreatitis and are used in various critical care settings whereas Ranson’s criteria is specifically designed for acute pancreatitis. e) Though BISAP score is simple & easy to use, it predicts based on the assessment within first 24 hours of admission. It is relatively inferior in predicting the complications or mortality which can happen later in the course of illness. Ranson’s score is relatively superior to BISAP in this context as it considers assessment both at admission & 48hours after admission. f) APACHE II is more complex, need more data to calculate and it is not specific to acute pancreatitis as compared to Ransons score. why not Atlanta Classification? Atlanta classification is based on morphological assessment of acute pancreatitis severity on CT which usually takes places after atleast 48-72 hours. based on that we give CSI & MCSI scores. Till then Ranson’s score will provide early prognostic information before detailed imaging findings are available. Method: 1. How was pancreatitis diagnosed? The diagnosis of pancreatitis in our study followed a rigorous, multi-dimensional approach in line with established clinical guidelines. Initially, we considered the patient’s clinical presentation, including relevant history (e.g., alcohol consumption, gallstones), as well as symptoms like abdominal pain and vomiting. Objective findings such as ileus, fever, tachycardia, and hypotension were also critical. Crucially, we substantiated our clinical suspicion through laboratory evidence, particularly elevated lipase and amylase levels, which were at least threefold above the upper limit of normal. Finally, imaging played a pivotal role in our diagnostic algorithm. Contrast-Enhanced Computed Tomography (CECT) of the abdomen or Ultrasound was utilized to confirm the diagnosis, adhering to the principle that the clinician’s discretion, based on a synthesis of these data points, ultimately guided the diagnostic process. 2. Institutional protocol for managing pancreatitis At our institution, the Department of Surgery manages pancreatitis cases. The management protocol begins at the point of presentation, where patients with symptoms indicative of pancreatitis or those evaluated for abdominal pain are assessed by the attending surgeon. Our approach prioritizes conservative management, including bowel rest, fluid resuscitation, and the administration of IV antibiotics and analgesics. Supportive measures such as supplemental oxygen, inotropic or ventilatory support, and transfusions for DIC are employed based on individual patient needs. Admission decisions—whether to the ward for mild cases or the ICU for those with significant organ dysfunction or unstable vitals—are made in accordance with the severity of the clinical presentation. Follow-up and further management, including imaging with CECT abdomen, are guided by established criteria, such as Ranson's criteria, and the clinical judgment concerning suspected complications. Reference : (Banks PA, Freeman ML. Practice guidelines in acute pancreatitis. Am J Gastroenterol. 2006;101:2379–2400.) 3. At what stage of disease was CT done? The timing for deploying CECT in our cases was carefully considered, primarily focusing on patients exhibiting signs of complicated pancreatitis, as defined by the Atlanta criteria, or when clinical suspicion of complications arose. Adhering to best practice, we generally deferred CT imaging for at least 48-72 hours following the onset of symptoms. This delay allows the acute inflammatory processes to stabilize, thereby enhancing diagnostic accuracy and informing subsequent management decisions, particularly in our ICU patients who are closely monitored for any signs of worsening or complications. Reference : Busireddy KK, AlObaidy M, Ramalho M, Kalubowila J, Baodong L, Santagostino I, Semelka RC. Pancreatitis-imaging approach. World J Gastrointest Pathophysiol. 2014 Aug 15;5(3):252-70. doi: 10.4291/wjgp.v5.i3.252. PMID: 25133027; PMCID: PMC4133524.) 4) Do you perform all investigations as required to calculate Ranson’s criteria on all patients with AP? The investigations for Ranson's criteria were not performed for all cases but based on the clinical condition as per the discretion of the attending surgeon. Complete blood counts, glucose, calcium, liver and renal function tests and blood gas was performed at baseline for all cases whereas the post 48 hour investigations were done selectively based on the clinical condition of the patient. Based on the predicted severity, a decision to perform a CECT scan abdomen was taken. 5. What do you mean by critical care? What is your institute’s protocol in admitting the patients for critical care? We had a Surgical intensive care unit at our hospital with ventilators and routine intensive care support systems. Unfortunately, there was no step-up or high dependency unit (HDU) facility available. Any patient who was found to have any organ dysfunction or features of shock was admitted to the ICU for monitoring and treatment and started on appropriate treatment. They were provided with supplemental oxygen, Non invasive ventilation (NIV) or endotracheal intubation with ventilation as and when required and hemodynamic support with transfusions or inotropic and pressor supports along with routine conservative management. Discussion: 6) unique in our study : There was no other study in the literature comparing both radiological severity indicators CSI & MCSI with Ranson's score alone in this combination at the time of conceptualization of this study. there were studies comparing CSI alone or MCSI alone with Ranson score or correlating radiological scores (CSI/MCSI) with multiple clinical scoring systems. 7)Limitations of study : a) the patients who underwent CECT for acute pancreatitis but who were discharged against medial advice or lost for follow up could not be assessed and excluded from the study. b)the study did not include patients below 12 years of age as Ranson's criteria is not done for paediatric patients in our hospital. c)smaller sample size which may increase the margin of error when compared to a study with a relatively larger sample size. d) Since no single scoring system can universally predict the patient outcome perfectly, it is a limitation of the study. e) Ranson's score needs 48hours to complete the score. CSI & MCSI is obtained after atleast 48-72hours of admission. So there is delay of obtaining scores and hence it has lesser sensitivity in very early stages of disease. We trust these responses address your queries comprehensively. Our team is committed to delivering research that meets the highest standards of clinical rigor and transparency. Should you require further details or clarification, we are at your disposal." } ] }, { "id": "206377", "date": "29 Sep 2023", "name": "Jan Trna", "expertise": [ "Reviewer Expertise gastroenterology", "endoscopy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting topic with interesting results showing that CT results can predict severity well. This is probably not a completely new finding, however comparison with Ranson scoring system and showing that CTSI is better is interesting since CT is reproducible and not subjective. There are many new scoring systems and showing that good old CTSI is good is in my opinion a very good piece of evidence.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "11284", "date": "04 Apr 2024", "name": "Griselda Noronha", "role": "Author Response", "response": "Thank you so much for your valuable comments and review report. Highly appreciate your feedback." } ] }, { "id": "206452", "date": "29 Sep 2023", "name": "Juan Xiao", "expertise": [ "Reviewer Expertise Biochemistry", "baic medicine，acute pancreatitis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 described the usefulness of modified CT severity index in the prediction of acute pancreatitis severity and outcome which is of clinical significance. However, there are still some issues which need to be addressed.\nThe authors should put the MSCI and SCI results compared to the same parameter together in one figure but not separately in Figure 1-5, 7.\n\nThe Ranson score result should be added in figure 6 and 9 in order to support your conclusion that MSCI is better than Ranscon score in outcome prediction.\n\nDid the MSCI score correlate to the etiologies of acute pancreatitis?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11285", "date": "04 Apr 2024", "name": "Griselda Noronha", "role": "Author Response", "response": "Dear Reviewer, Thank you for your detailed review of our article titled \"Comparative Analysis of Computed Tomography Severity Indices in Predicting the Severity and Clinical Outcome in Patients with Acute Pancreatitis\" submitted to the F1000 Journal. We appreciate your time and effort in providing constructive feedback to enhance the quality of our research. We acknowledge your suggestions. 1) Based on your feedback, we will revise the figures accordingly to incorporate the MSCI and CSI results compared to the same parameter together in one figure but not separately in Figure 1-5, 7. We are committed to addressing all your concerns and making the necessary revisions to strengthen the validity and impact of our findings. Your valuable input will undoubtedly improve the clarity and reliability of our study, and we are grateful for your insightful suggestions. 2) Regarding the addition of the Ranson score in Figures 6 and 9 of our manuscript to better support the conclusion that the MCSI is superior to the Ranson score in predicting clinical outcomes in patients with acute pancreatitis, we apologize for any confusion or oversight on our part in not including the Ranson score in the mentioned figures. It would be great if you could suggest how to incorporate the Ranson score in figure 6 and 9 as we are unable to figure it out. I discussed with my statistician and he was unable to figure it out. Hence your input would be greatly appreciated on this. 3) In our study we found that the most common etiology for acute pancreatitis was alcoholism followed by gall stones and third was idiopathic. we have not correlated MCSI with acute pancreatitis etiologies in our study. We will promptly update the manuscript as per your recommendations and hope that these revisions will further enhance the significance and relevance of our research in the field of acute pancreatitis management. Thank you once again for your thorough review and for guiding us to refine our work for publication in F1000 Journal. Sincerely, Griselda Philomena Noronha Corresponding Author" } ] } ]	1	https://f1000research.com/articles/11-1272
https://f1000research.com/articles/12-1306/v1	11 Oct 23	{ "type": "Clinical Practice Article", "title": "Few incidentally found interesting foreign objects in human body: a case series", "authors": [ "ANAND HATGAONKAR", "KAJAL HATGAONKAR", "SANDEEP DHOTE", "VAISHALI DHAWAN", "ANAND HATGAONKAR", "SANDEEP DHOTE", "VAISHALI DHAWAN" ], "abstract": "Foreign bodies are objects that do not typically belong in the human body but can be ingested, inserted, or entered due to injuries. This article presents various cases and examples of foreign bodies, including objects swallowed, objects inserted into the rectum, vagina, urethra, ear, and nose, or due to injuries caused by falls, puncture wounds, and gunshot wounds. Foreign bodies can be difficult to detect, particularly if they are not inherently radio-opaque, and may be overlooked by patients who cannot provide an adequate history. These foreign bodies may cause harm to the patient. Interpretation is done on radiographs, computed tomography (CT), Ultrasonography (USG), and magnetic resonance imaging (MRI) imaging studies. Most foreign objects pass through the gastrointestinal tract without problem; sharp and elongated objects can cause significant injury, and even if they only partially perforate the bowel wall, they can produce chronic inflammatory processes that produce symptoms months or years later. Hence, searching for foreign bodies should be done throughout the gastrointestinal tract, particularly in children and people with mental illness who are more likely to swallow multiple items more than once. Although rare, various materials can be left behind in the body of a patient after surgery, including large and small wire sutures, surgical drains, and retained sponges, which can cause potential complications and foreign body reactions. This article highlights the importance of being aware of the presence of foreign bodies in clinical practice, and a thorough search should be carried out using different modalities, especially CT. Great suspicion and early diagnosis of foreign bodies can avoid potential complications and morbidity. In general, it provides information on the diagnosis and treatment of various types of foreign bodies.", "keywords": [ "foreign bodies", "ingestions", "insertions", "injuries", "X-ray", "CT scan", "ultrasound", "MRI." ], "content": "Introduction\n\nThe human body is and forever will be an amazing mystery. But sometimes it is even more surprising to find things that do not normally belong in a human body, like a pen refill in the stomach or a sharp metallic object in the bladder. Although they are rare, foreign bodies are fascinating and significant. They can be overlooked and can cause harm to the patient. If one does not suspect the presence of a foreign body, interpreting radiograph, computed tomography (CT), ultrasonography (USG), and magnetic resonance imaging (MRI) investigations are especially prone to inaccuracy.1 Children, mentally challenged people, adults who exhibit atypical sexual behavior and even “normal” adults or children with risk factors are more likely to consume or introduce foreign bodies.2\n\nThis article discusses key concepts about foreign body ingestions, insertions, and injuries while illustrating a range of shocking foreign bodies.\n\nMethods: This case series was carried out at tertiary care centre in central India. Radiography was done on digital and computerized radiography X-ray machine; multi-slice CT scanner and 1.5 Tesla MRI.\n\nEthical consideration: All ethical principles were followed during the study and all measures are taken to maintain anonymity. Institutional ethical committee of Shalinitai Meghe Hospital and research center, which is constituent unit of Datta Meghe Medical College have granted ethical clearance for study vide letter no. SMHRC/IEC/2023/02-59 dated 17/02/2023.\n\nConsent: Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/relative of the patient.\n\n\nCases\n\nCase 1: A four-year-old child came with a history of abdominal pain. On a radiograph of the abdomen, frontal and lateral projections reveal circular radio opacity on the left side at the level of the L2-L3 disc suggestive of coin, which the patient had accidentally ingested. It passed through the gastro-intestinal tract without a problem.\n\nCase 2: A 34-year-old male carpenter by profession accidentally ingested a screw. A radiograph of the abdomen in frontal and lateral views reveals a nail at the level of the L4 and L5 vertebrae in the gastrointestinal tract, which passed without any problems.\n\nCase 3: A 25-year-old male patient came for ultrasound examination with complaints of pain in the abdomen for two-three months. Radiograph of the chest and abdomen reveal multiple linear radio-opacities in the left hypochondriac and lumbar quadrants of the abdomen, and a plain CT scan shows multiple hyperdense linear metallic foreign bodies within the gastric lumen, many piercing the gastric wall partially without any evidence of perforation. On laparoscopy, multiple refills of the pen and wires were found in the stomach, which were removed.\n\nCase 4: An 18-year-old woman patient came with a history of abdominal pain and vomiting on and off for 15 days. Contrast enhanced computed tomography (CECT) reveals a heterogeneous lamellated non-enhancing soft tissue density mass (with a wide attenuation range from -70 to 70 HU) intraluminally in the stomach, conforming to its shape and extending into the antrum, pylorus, and minimally into the duodenal cap suggestive of trichobezoar. Gastrotomy revealed the ball of hair in the stomach.\n\nCase 5: A seven-year-old boy came with a history of epistaxis. CT paranasal sinuses (PNS) revealed a non-enhancing hyperdense lesion in the left nasal cavity, possibly a foreign body. It was removed under anaesthesia and found to be a castor seed.\n\nCase 6: A 17-year-old woman came for an ultrasound examination in emergency hours with complaints of severe pain in her lower abdomen. Radiograph Pelvis anteroposterior (AP) view revealed long radio-opacity in the bladder with a radiolucent center that did not look like a calculus, but a foreign body. USG revealed a linear hyperechoic foreign body that penetrated the anterior wall of the bladder. The patient had a history that she had conceived three years ago and had tried abortion by a quack in her village. The patient was operated on and a shaggy piece of a long wooden stick with cotton wrapped around it was found.\n\nCase 7: A 40-year-old male presented with a complaint of pain in the right heel region for two months. A radiograph of the lateral view of the right foot revealed there was evidence of calcific tendinitis of the Achilles tendon with thickening of the Kager fat pad and fat stranding. USG revealed that a well-defined thorn visualized in the Achilles tendon with associated surrounding tendinitis and increased fat echogenicity. USG-guided thorn removal was performed.\n\nCase 8: A 50-year-old woman came for cervical spine. The patient was taken for an MRI scan when she complained of a severe headache. Radiograph skull AP & Lateral view was done, which showed a radiodense nail-like structure in scalp on right side. On asking, the patient gave a history of trauma ten years back and did not know that she had a nail in her scalp. It was removed under local anaesthesia.\n\nCase 9: A 37-year-old man had a history of bullet injuries. Radiographs of the chest and abdomen in frontal and lateral views revealed multiple pellets in the subcutaneous soft tissue of the thorax and abdomen.\n\nCase 10: A 33-year-old female came with a history of bleeding pervaginal for six months. MRI shows heterogeneous altered signal intensity soft tissue mass anterior to the uterus with multiple hypointense foci within. CT showed multiple linear metallic strings within a mass of soft tissue density anterior to the uterus, suggesting a foreign body (gossypiboma). The patient was operated on, and a large surgical sponge was removed.\n\n\nResult\n\nA total of ten cases were studied, comprising of four females and six males of various age groups ranging from four years to 50 years (Tables 1 & 2). Four patients had ingested foreign bodies while two patients had history of insertion and four other had insertion due to injury (Table 3).\n\n\nDiscussion\n\nForeign bodies are objects that do not typically belong in the human body but can be ingested, inserted, or entered due to injuries. This article presents various cases and examples of foreign bodies, including objects swallowed, objects inserted into the rectum, vagina, urethra, ear, and nose, or due to injuries caused by falls, puncture wounds, and gunshot wounds.\n\nThe swallowing of foreign bodies is a common condition in children and mentally challenged individuals.3–5 Fortunately, most ingested objects move through the digestive system without causing any problems (Figure 1a,b). Sharp and elongated objects can pass uneventfully (Figure 2a,b); however, they can pierce the mucosal lining and seriously damage or completely perforate the intestinal wall (Figure 3a-e). The object may just partially puncture the gut wall, resulting in a chronic inflammatory condition with few symptoms that is diagnosed months or years later.5–7\n\nOn laparoscopy, multiple refills of the pen and wires were found in the stomach, which were removed (e).\n\nWhen a patient cannot provide a sufficient history or has swallowed things that are not naturally radio-opaque, the diagnosis of an ingested foreign body is frequently missed. If a foreign body is suspected and is not visible on a Radiograph because of its radiolucent nature, a CT scan of the abdomen or chest may be beneficial8 (Figure 4a,b).\n\nGastrotomy revealed the ball of hair in the stomach (b).\n\nSometimes you may not have a proper history of the ingestion of sharp objects. When a patient has a history of ingesting a foreign body, whether it is an adult or a kid, they should be checked for the entire body, from the base of the skull to the anus, from the nasopharynx to the rectum. The hunt for other foreign bodies should not stop just because one has been discovered because youngsters are particularly prone to eating items in multiples.9\n\nThe rectum, vagina, urethra, ear, and nose are common places for foreign items to be inserted. These are especially common in children (Figure 5a,b) but can also be seen in adults. The deposition of mineral salts is especially likely to occur in foreign bladder substances, resulting in the formation of bladder calculi (Figure 6a,c). In fact, when a child or young adult develops a bladder calculus, the presence of an embedded foreign body should be suspected.10\n\nUSG revealed a linear hyperechoic foreign body that penetrated the anterior wall of the bladder (b). The removed foreign body was a long wooden stick with cotton wrapped around it (c).\n\nMost people may have experienced at least one or two minor injury incidents, such as falls, abrasions, cuts, scrapes, and burns. Few of them may have experienced injuries from firearms and may have experienced puncture wounds from splinters, thorns, needles, or glass.2\n\nOn ultrasound, all foreign bodies in soft tissue are initially hyperechoic. Sonography is important for the correct localization of all kinds of soft tissue foreign bodies and the detection of non-radiopaque foreign bodies. Accurate localization can help minimize surgical exploration and can also direct the percutaneous removal of a foreign body11 (Figure 7a,b)\n\nUSG revealed that a well-defined thorn visualized in the Achilles tendon with associated changes of tendinitis (b). Thorn removed under ultrasound guidance (c).\n\nSome metallic foreign bodies can be accidentally diagnosed during an MRI or CT study due to artefacts or sometimes due to pain as they enter the magnetic field12 (Figure 8a,b).\n\nThe nail removed under ultrasound guidance (c). Normal radiograph skull A-P & Lateral view post removal of the foreign body (d,e).\n\nThe gauge of a shotgun pellet determines its size, the higher the number, the smaller the pellet. Serious soft tissue and bone damage can result from the combined mass striking a target close to the gun barrel (Figure 9a-d). Because steel pellets are ferromagnetic, they could move dangerously if such a patient with embedded steel pellets was exposed to a magnetic field, making magnetic resonance imaging potentially dangerous.2\n\nAfter surgery, not infrequently, patients have surgical items inside their bodies. Surgical drains, wound gauze packs, bandages, skin staples, small surgical staples, intra-arterial, intravenous, intra-spinal, and intraabdominal catheters are among the postoperative supplies that are most frequently seen. Other uncommon materials, such as retained abdominal sponges (Figure 10a-d) and needles, that were unintentionally left behind after surgery, are challenging to find clinically and radiographically because patients have vague symptoms, these objects are difficult to see on radiographs, and the radiologist and referring physician have a low level of suspicion for such objects. The nursing staff may perform a comprehensive sponge count at the conclusion of a surgical procedure and identify any remaining surgical sponges right away. A misplaced sponge may not be identified for months or even years after surgery if it is not found at that time. The foreign body reaction to a surgical sponge left inside the body for a long time is frequently called a gossypiboma. The sponge’s cotton matrix is what creates the foreign body reaction’s nidus. There is the development of a foreign body granuloma with surrounding fibrosis and retraction around the cotton nidus. Many people have no symptoms, and the retained sponge is often only unintentionally found when the patient has a radiological examination for another reason.13–17\n\n\nConclusion\n\nForeign bodies are interesting, and most of them are diagnosed incidentally in various parts of the human body and can cause significant harm if not properly managed. The diagnosis and management of foreign bodies can be challenging and require a high index of suspicion. Imaging studies such as Radiographs, CT scans, USG, and magnetic resonance imaging can be helpful in detecting and localizing foreign bodies. The management of foreign bodies can involve a variety of interventions, including endoscopy, surgical exploration, and percutaneous removal. Prevention is also key, particularly in children and mentally handicapped adults who are at increased risk of foreign body ingestion or insertion. It is important for healthcare providers to be aware of the potential for foreign bodies and to maintain a high level of vigilance when evaluating patients. Ultimately, early detection and appropriate management can prevent serious complications and improve patient outcomes.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nCARE guidelines for case reports: 13-item checklist\n\n\nReferences\n\nCarneiro BC, Cruz IA, Chemin RN, et al.: Multimodality imaging of foreign bodies: new insights into old challenges. Radiographics. 2020 Nov; 40(7): 1965–1986. PubMed Abstract \| Publisher Full Text\n\nHunter TB, Taljanovic MS: Foreign bodies. Radiographics. 2003 May; 23(3): 731–757. Publisher Full Text\n\nLee JH: Foreign Body Ingestion in Children. ClinEndosc. 2018; 51(2): 129–136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOliva S, Romano C, De Angelis P, et al.: Foreign body and caustic ingestions in children: A clinical practice guideline. Dig. Liver Dis. 2020; 52(11): 1266–1281. PubMed Abstract \| Publisher Full Text\n\nYıldız İ, Koca YS, Avşar G, et al.: Tendency to Ingest Foreign Bodies in Mentally Retarded Patients: A Case with Ileal Perforation Caused by the Ingestion of a Teaspoon. Case Rep. Surg. 2016; 2016: 8075432–8075433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFleisher AG, Holgersen LO, Stanley-Brown EG, et al.: Prolonged gastric retention of a swallowed coin following pyloromyotomy. J. PediatrGastroenterolNutr. 1986; 5(5): 811–813. Publisher Full Text\n\nMartel G, Johnston D, Jones C, et al.: Liver perforation following foreign body ingestion: an important clinical lesson. BMJ Case Rep. 2015 Jul 7; 2015: bcr2015210098. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIwama I: Overlooked radiographic finding results in delayed diagnosis of a retained oesophageal foreign body. BMJ Case Rep. 2014; 2014: bcr2014204856. Published 2014 Aug 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConners GP, Mohseni M: Pediatric Foreign Body Ingestion. [Updated 2023 Jan 1]. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 Jan. Reference Source\n\nUnruh BT, Nejad SH, Stern TW, et al.: Insertion of foreign bodies (polyembolokoilamania): underpinnings and management strategies. Prim Care Companion CNS Disord. 2012; 14(1): PCC.11f01192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorton LK, Jacobson JA, Powell A, et al.: Sonography and radiography of soft-tissue foreign bodies. Am. J. Roentgenol. 2001 May; 176(5): 1155–1159. Publisher Full Text\n\nVoss JO, Maier C, Wüster J, et al.: Imaging foreign bodies in head and neck trauma: a pictorial review. Insights Imaging. 2021 Dec; 12(1): 1–6. Publisher Full Text\n\nDhillon JS, Park A: Transmural migration of a retained laparotomy sponge. Am. Surg. 2002 Jul; 68(7): 603–605. PubMed Abstract \| Publisher Full Text\n\nJason RS, Chisolm A, Lubetsky HW: Retained surgical sponge simulating a pancreatic mass. J. Natl. Med. Assoc. 1979 May; 71(5): 501–503. PubMed Abstract\n\nRappaport W, Haynes K: The retained surgical sponge following intra-abdominal surgery. Arch. Surg. 1990; 125: 405–407. Publisher Full Text\n\nGümüş M, Gümüş H, Kapan M, et al.: A serious medicolegal problem after surgery: Gossypiboma. Am. J. Forensic Med. Pathol. 2012; 33(1): 54–57. PubMed Abstract \| Publisher Full Text\n\nSilva CS, Caetano MR, Silva EA, et al.: Complete migration of retained surgical sponge into ileum without sign of open intestinal wall. Arch. Gynecol. Obstet. 2001 May; 265: 103–104. PubMed Abstract \| Publisher Full Text" }	[ { "id": "237799", "date": "20 May 2024", "name": "Kai Sheng Hsieh", "expertise": [ "Reviewer Expertise Pediatrics", "Pediatric Cardiology", "Pediatric Emergency-Critical Care", "Pediatric Pulmonology", "Neonatology", "Perinatology", "Ultrasound/Echocardiography" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article entitled \"Few incidentally found interesting foreign objects in human body: a case series\" was reviewed. The article summarized 10 cases of different forms of foreign body retention within the human body . Their ages ranged from 4 years old to 50 years old. Multiple modalities of diagnostic equipment were employed for imaging purpose. With these remarkable experience, the authors conclude that physicians should be alerted about the possibility of foreign body retention within the human body. Although the article is well written, I have the following comments for authors to respond: 1. Please add the duration of symptom(s) for each individual case. Most of the 10 cases lack this data (the information is present only in cases 3,4,7 and 10) . 2. Please add the concise essential findings of physical examination (significant positive nd negative findings). 3. Please add some sentences about the choices of diagnostic imaging ( Plain X-rays, CT , MRI and ultrasound) in the Discussion section. 4. Please add some sentences regarding the analysis of “risk factors” including those present in this article and those mentioned in other literatures in the Discussion section.\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [ { "c_id": "11860", "date": "28 Jun 2024", "name": "ANAND HATGAONKAR", "role": "Author Response", "response": "Dear Kai Sheng Hsieh, Thanks for the review, I am really thankful to you for nice and thoughtful review. Your review had helped me in making this article more comprehensive. As it is all about imaging I have added all necessary details about it. Thanks & Regards, Dr. Anand Hatgaonkar" } ] }, { "id": "278383", "date": "30 May 2024", "name": "Giacomo Calini", "expertise": [ "Reviewer Expertise surgery", "endoscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 praise the authors for this interesting and broad case series and review of novel and rare episodes of foreign objects.\n\nIn the case of high-risk ingested foreign bodies endoscopy is required. Therefore, my advice is to discuss this treatment option, including the publications below (Ref 1-3).\nAlso, terms like mentally handicapped, are outdated in medicine as they generally carry an offensive intent. Revise these term with a more appropriate terminology. eg, people with intellectual disability\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? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [ { "c_id": "11869", "date": "25 Jun 2024", "name": "ANAND HATGAONKAR", "role": "Author Response", "response": "Thanks for the review, As per your suggestion, we have updated information about the treatment strategies used to remove different foreign objects from human body." } ] } ]	1	https://f1000research.com/articles/12-1306
https://f1000research.com/articles/13-811/v1	17 Jul 24	{ "type": "Research Article", "title": "Floristic Diversity of Natural Forest Patches Across Agroclimatic Zones in Northwestern Ethiopian", "authors": [ "Melkamu Kassaye", "Yonas Derebe", "Mulugeta Tamer", "Amsalu Nigatu", "Bahiru Gedamu", "Estegenet Emiru", "Meseret Chanie", "Mebratu Yigzaw", "Yonas Derebe", "Mulugeta Tamer", "Amsalu Nigatu", "Bahiru Gedamu", "Estegenet Emiru", "Meseret Chanie", "Mebratu Yigzaw" ], "abstract": "Background Natural forest resources in northwestern Ethiopia are currently under threat, requiring immediate conservation measures and a management strategy. Thus, an understanding of the present floristic diversity status is required. There is lack of such type of studies specific to fragmented forest patches and across environmental factors. Consequently, this study aimed to examine the existing state of these resources to apply sustainable forest management practices.\n\nMethods To achieve this, transects with 200 square plots measuring 400 m2 were set up at nine forest patches with 200m spacing between plots and transects. Vegetation and environmental data were collected and analyzed using R version 4.1.\n\nResults Significant variations (p 0.008) were found in vegetation features along the slope, forest patches, and agroclimatic zone. When compared to other forest patches, highland, and mid-altitude forest patches had the highest species diversity (2.48) and stocking (2578 trees/ha). The gentle slope has the most species diversity (2.83). The species similarity between highland and mid-altitude forest habitats was found significant (69%). The vegetation cover in the mid-altitude forest patches was also high (abundance: 5-12%). In all the forest patches examined, tree life forms exceeded shrubs, climbers, and herbs.\n\nConclusions According to the study’s findings, the state of forest resources varies considerably across different environmental variations. Despite the impression of entire forest patches from the outside, the interiors are open, with only huge and mature trees covering the canopy. This state has been triggered by deforestation, degradation, and inappropriate human and grazing operations.", "keywords": [ "floristic diversity", "vegetation structure", "vegetation distribution", "forest patches", "environmental factors" ], "content": "Introduction\n\nForest vegetation is an important natural resource with global ecological, economic, and social implications. Forest vegetation is an important component of the biogeochemical cycle and aids in the protection of the earth from natural and anthropogenic risks, particularly in moderating climate change. Many economic lifestyles rely on trees and shrubs for food, timber, medicinal supplies, and textiles. Religious and cultural activities depend heavily on forest resources. As a result, forest vegetation is critical to sustaining the global, national, and local balance of nature (Coomes et al., 2016). However, due to deforestation and overuse, these vital resources are dwindling. Urgent resource valuation according to site features is required for conservation and long-term use (Nzabarinda et al., 2021).\n\nThe interaction of living and non-living elements influences the dynamics of forest ecosystem vegetation (Cormack et al., 1979; Kent, 2012; Franklin, 2013). The principal sources of temporal and spatial changes in vegetation are natural plant development processes and disturbances (Hull et al., 2018). Anthropogenic and natural disturbances have a significant impact on forest ecosystem vegetation, with time and place variations. Time is considered to be a key factor in vegetation dynamics (Cormack et al., 1979). Rainfall, temperature, wind, light, humidity, and other climatic variables change throughout time and influence ecological variables that affect plant ecology and evolution. Furthermore, changes in population development contribute to a rise in anthropogenic disturbances throughout time. The interactions of natural and manmade disturbances cause ongoing vegetation dynamics within the forest ecosystem (Tuxen, 1974; Cormack et al., 1979; Franklin, 2013; Hull et al., 2018; Sales et al., 2019).\n\nForest vegetation is extremely variable in terms of structure, composition, growth morphology, and geographical distribution (Kent, 2012; Hull et al., 2018). Vegetation dynamics are influenced by environmental elements such as slope, elevation, aspect, and site conditions (Dwyer et al., 2022). The degree of differences in vegetation studies, however, differs depending on whether they are conducted at the biome, ecosystem, community, or population level (Musau et al., 2016). There are variations in structure and growing conditions even within the same species of vegetation. Primary features that fluctuate in place and time are species makeup, structure, and growth performance (Belay et al., 2013; Jyothi and Sureshkumar, 2018; Aide et al., 2019; Angessa et al., 2020; Dang and Trung, 2021). These parameters can be used to measure vegetation dynamics (Pease et al., 2022). Examining vegetation dynamics across different environmental patterns is critical for sustainable forest management and utilization. It also supports the design and administration of ecological restorations and management by identifying environmental factors that influence the spatial distribution and quantity of vegetation (Kent, 2012; Gemedo et al. 2014).\n\nEthiopia has a wide range of agroecological zones, from the top Ras-Dejen Mount to the lower Afar Depression, which results in a wide range of plant species typical of tropical climates (Bekele, 2007). However, human activities such as deforestation, agricultural land development, overgrazing, high demand for firewood and charcoal, and a lack of adequate legislative frameworks are causing alarming degradation of flora resources (Soromessa et al., 2004). Conservation actions, particularly in the study areas, are urgently needed to protect these Afromontane forests (Senbeta et al., 2014). Effective conservation strategies that assure long-term viability are required. Unfortunately, there have been very few scientific studies on forest vegetation distribution, diversity, and structure, as well as growth performances in relation to natural and anthropogenic causes (Caroline et al., 2020), particularly in the Awi zone, northwest Ethiopia.\n\nAs a result, the purpose of this investigation is to evaluate the diversity, composition, distribution, and growth performance of forest vegetation in relation to environmental circumstances. The study also investigates the impacts of slope, aspect, altitude, and agroclimatic zone on vegetation diversity, structure, and composition, while considering the interaction of human and livestock disturbances. The study gives important scientific knowledge for the protection and long-term management of natural forest vegetation. It also provides an initial for policymakers to use when developing and implementing forest restoration, environmental management, and climate change mitigation strategies.\n\n\nMethods\n\nThe study was carried out in the Amhara region’s Awi administrative zone, which is located between 11° to 10°85’N latitude and 36°39’60” to 36°57’E longitude. The study area has an altitudinal range of 600 to 3500 m. a. sl., 1750 mean annual rainfall, and temperatures ranging from 170°C to 27°C. Awi (Agew-mider) has a rather level topography (Figure 1).\n\nAgroecologically, most of the Awi administrative Zone is highland (17%), mid-altitude (72%), and lowland (11%). The rainfall distribution in the area is ideal. Geographically, the Awi zone has a total area of 8,935,520 ha of land, of which 297,133 ha (33.25%) is agricultural land and 354,396 ha (34.02%) are forest land (76,554 ha plantation and 277,842 ha natural forest). The remaining land is rangeland and grazing land, covering 217,138 ha (24.3%), and other land uses, including as infrastructure and settlement, covering 74,853 ha (8.38%) (Awi Zone Agricultural office, 2021).\n\nAgew-mider (Awi) has a total population of 1,159,386. The urban population accounts for 13% of the total population; the rest live in rural areas and rely on mixed farming lifestyles (Genet, 2022). Currently, the people in the highlands parts practices Acacia mearnsii forest farming in the Taungya farming system. The small-scale Acacia mearnsii woodlot is responsible for reclaiming acidic soil and enhancing soil fertility, but its major function is to generate money through charcoal manufacture (Wondie and Mekuria, 2018). One of the studied districts, Jawi, has a significant potential for natural forest cover (48.9%), followed by Guangua (20.5%) and Ankesha (8.27%).\n\nHighland (2200-3600 m.ab.sl.), Mid-altitude (1300-2200 m.ab.sl.), and Lowland (500-1300 m.ab.sl.) were the primary Awi Zone classifications (Bekele, 2007; Gorfu and Ahmed, 2011). One or two districts in each agroclimatic zone were purposely chosen based on the potential for natural forest cover. To obtain sufficient biophysical data, three forest patches with high area coverage were chosen in each target district and agroclimatic zone (Alemayehu, 2007). Systematic sampling was used to acquire biophysical data in the forest under each designated forest patch. Then, using GPS and GIS tools, transects and plots were put out based on area coverage. The transect and plots were 200 meters apart. Transects were laid across the elevation gradient, followed by transects and then plots. The main plot was 20m by 20m for trees, with two 10m by 10m sapling subplots, five 5m by 5m seedling and regeneration subplots, and five 1m by 1m herbaceous species subplots within the main plots. The number of plots investigated in a particular forest varies according to the species-area curve (Kent, 2012).\n\nTo achieve this, vegetation and environmental data from nine forest patches in 72 plots were collected. Following standard inventory procedures, tree growth parameters (height and DBH) were measured with a hypsometer and diameter tape, respectively, and the life form of a species was clustered as trees (DBH > 2.5 cm and height of >2.5 m), saplings (DBH 2.5 cm and height of 1m h 2 m), and seedlings and herbs (DBH 2.5 cm and height of 1 m) at each plot (FAO, 2004; Kent, 2012). Site parameters such as slope class (gentle slope = 1-10%, mid-slope = 10.1-20%, and upper slope = 20.1-30%), aspect angle data were collected at each plot and classified as Northeast (NE), Northwest (NW), Southeast (SE), and Southwest (SW), and elevation were classified for individual forest patches were collected using GPS. The species-area-curve model was used to determine the optimal number of plots (FAO, 2006).\n\nThe vegetation characteristics were computed and compared with biodiversity indices such as the Shannon Weiner diversity index (H’), Sorenson’s similarity index (Ss), and Shannon Weiner evenness index (SEI) (Gotelli & Anne, 2013; Naidu and Kumar, 2016).\n\nThe species diversity index with the Shannon-Wiener index:\n\nWhere: H’ = Diversity of species S = Number of species Pi = the proportion of individuals abundance of the ith species\n\nSorenson’s similarity Coefficient (Ss)\n\nWhere: a = number of species common to both habitats; b = number of species present in the first habitat but absent in the second; c = number of species present in the second habitat but absent in the first.\n\nShannon Weiner evenness index (SEI)\n\nWhere: H′: Shannon Weiner diversity index, S: number of species\n\nImportant value index (IVI)\n\nWhere: IVI = RDe = Relative density, (RDe) + RDo = relative dominance, (RDo) + RF = relative\n\nFrequency\n\nForest species were identified using NDA (Natural Database Africa) software Ver. 2.0 and the Flora of Ethiopia and Eritrea book (Bekele, 2007), vegetation descriptions, and environmental patterns were analyzed using Kent (2012), Zhang et al. (2013), Lemessa et al. (2017) methods. Brawn Banquets cover analysis was used to determine vegetation cover: 1% extinct or uncommon, 1-5% bare or sporadic, 6-25% rare, 26-50% patches, 50-75% interrupted, >75% continuous. The connection of vegetation traits with environmental gradients and plot distribution was determined using principal component analysis (PCA) (Kent, 2012). All statistical studies (multivariate analysis, ANOVA, and cluster analysis) were carried out using different packages of R. Ver. 4.1.1 and PAST.Vr.3.\n\n\nResult and discussion\n\nThe possibility of obtaining new species increases up to 2.4 hectares, beyond which the species richness becomes saturated. This means that species recruitment was possible up to a 2.4 ha plot area, and then the types of species recorded afterward were repetitive in highland and mid-altitude forest patches, whereas in lowland forest patches, the possibility of getting new species saturates at a 0.6 ha plot area (Figure 2).\n\nNote: Highland Forest patches: Darkan, Gubel, and Saharakani; Mid-altitude forest patches: Den Maryam, Dukima, and Elala; Lowland Forest patches: Asech, Ambaser, and Abuhay Dengara.\n\nThe species-area curve is a species distribution model that illustrates the relationship between area and species richness with an indicator of the optimal number of plots for the inventory of floristic diversity in a specific forest patch (Gray et al., 2004). At 15 plots (0.6 ha), almost all forest patches had reached saturation in terms of new species.\n\nThere is no species variability after a certain elevation range in forest patches with substantial anthropogenic disturbances. Forest patches with small-scale spatial variability and significant disturbances have comparable species, whereas forest patches with vast spatial variability contain diverse species with even distribution, and the probability of discovering new species grows with the area. In some forest ecosystems, the possibility of acquiring new species extends until the last plots before becoming saturated. The environmental gradient in tropical Africa is highly variable, resulting in a wide range of species diversity in forest patches. This theory is comparable to that of Scheiner (2003) and Rosindell and Cornell (2009), who claim that floristic species are evenly distributed across the forest and that there are new species in every aspect of the forest patches.\n\nFloristic diversity\n\nFloristic diversity and species richness varied significantly (p 0.008) over slope gradients in different agroclimatic zone and forest patches. Mid-altitude forest patches had the highest floristic species richness (78 species in 32 Families) (Table 2). The Fabaceae and Rosaceae families account for 39% and 29% of the total species in mid-altitude and highland forest groups, respectively. Burseraceae, Combretaceae, and Fabaceae account for 75% of all species in lowland forests. Highland and mid-altitude forest patches exhibit higher floristic diversity (2.48) than lowland forest patches (1.88). The highest floristic diversity was found in the Dukima forest (2.83), followed by the Gubel forest (2.69) and Abuhay Dengara forest (2.49) (Table 2). The species diversity on the gentle slope is the highest (2.15), followed by the medium slope (1.96) and the steep slope (1.87) (Table 1).\n\nMid-altitude forests have more floristic diversity and richness. This could be the transition zone between lowland and highland forest patches. Because of the optimum climatic conditions, some lowland and highland species may favor mid-altitude areas. That is, vegetation characteristics, such as floristic diversity and species availability, have site-specific patterns, with some species preferring the extreme lower altitude of the highland and others preferring the extreme higher elevation of the lowland. This idea is expressed by Hawk et al. (1977), Franklin (2013), Gemedo et al. (2014), and Senbeta et al. (2014) who show that the species richness of plant patches changes reliably along environmental gradients. Another study by White and Hood (2004) stated that floristic diversity and distribution in forest patches are influenced by topographic and underlying features. Furthermore, according to Chetcuti et al. (2022) plant community distribution in landscapes is a manifestation of environmental gradients and biotic responses to these gradients.\n\nThe gentle slope is ideal for maximizing species diversity and richness. This could be because the lower areas of a forest are more suitable and productive for most floristic species. The finding supports Gemedo et al. (2014) argument that the higher richness of species diversity in gentle and flat slope areas of the forest is attributable to optimal environmental conditions for most species of life. Similar to the study of Franklin (2013), various environmental gradients and specific slopes have a direct influence on vegetation features, most notably diversity of species and richness. It is a natural law that environmental gradients and species compatibility matching regulate vegetation dynamics, specifically floristic diversity, and richness. Environmental gradients associated with natural and anthropogenic disturbance have a substantial impact on the geographical and temporal distribution of vegetation and life forms in an open natural forest. Each disturbance event, specifically in this study area—grazing and human impact—and the resulting site features distinguish a given forest patch.\n\nFloristic species similarity\n\nThere was significant positive species similarity (p 0.024) between and among mid-altitude and highland forest ecosystems. However, there were significant floristic species dissimilarities (p 0.001) between forest patches of highland and lowland areas. There was also significant species similarity within the lowland forest ecosystem (Figure 3).\n\n((A/Dengara = Abuhay Dengara, D/Maryam = Den Maryam); the red circle indicates a negative correlation; the blue circle indicates a positive correlation circle with X indicates a non-significant correlation (p > 0.05)).\n\nThere was a high degree of similarity (82% within lowland forest patches) and a medium degree of similarity (69% within highland forest patches). Highland and mid-altitude forests share 69% of their species presence; yet there are significant dissimilarities (68% between lowland and highland forest patches). There was also a 61% dissimilarity between mid-altitude and low-altitude forest patches.\n\nMid-altitude forest patches are more like highland and lowland forest patches, while the two extremes of highland and lowland forest patches are very different. Even while the degree of similarity or variability changes depending on a variety of anthropogenic and natural disturbances, the probability of species being similar within a single agroecological cluster is much higher (Della Rocca et al., 2021). The results of this study demonstrated that there was more than 50% similarity within the forest patches under the same agroclimatic zone, according to the Martín-Devasa et al. (2022) similarity index concept. Lowland forest patches, on the other hand, have unique species from highland and mid-altitude forest patches, suggesting a dissimilarity of more than 50%. According to Senbeta et al. (2014) and Lemessa et al. (2017), despite geographical distance, high floristic similarities were observed between patches located very close to each other, and dissimilar forest patches were expected if they had wide variations in altitudinal gradients and topographic aspects.\n\nFloristic species distribution\n\nSatellite species were identified in all elevation patterns at all forest patches, indicating that scattered species distribution with low abundance is prevalent. This revealed that there is significant deforestation and forest degradation, resulting in poor vegetation cover. Rural species distribution was similar in all elevation patterns, but the extent was less than that of satellite species. The distribution of urban species is widespread at lower and higher elevations when there is a patch of forests on the area’s hillsides. The distribution of core species was quite limited, with just a few species occurring at medium elevations where there is a transition zone and some management measures (Figure 4).\n\nAccording to the hierarchical continuum paradigm Kent (2012), species with high abundance and even distribution (core species) were only found in medium elevation gradients with optimal climatic and edaphic conditions. Satellite species were detected in all agroclimatic zone and elevation gradients with low abundance and inconsistent distribution. The species were uniformly distributed and scarce in higher elevation gradients, indicating that they are rural and satellite. A variety of species distribution types were discovered in the lower elevation gradient in the order satellite > rural > urban.\n\nFloristic species lifeform\n\nThere were a significant variation (p < 0.03) proportions among different lifeforms in the three agroclimatic zone forest patches. Tee components account for the lion’s share of all agroecological classes in each forest patch, with average contributions of 55%, 42%, and 67% in highland, mid-altitude, and lowland forest patches, respectively (Table 2).\n\nThe forest in the study area is an open natural forest that has been disturbed by anthropogenic factors. This is why lower plants become extensively degraded, and forest patches are made up of living forms with tree > shrub > climber > herb composition shares. This idea is comparable to that of Meragiaw et al. (2018) who found that tree and shrub life forms predominated in open natural forest patches. It is common for herbaceous species to experience grazing issues in the open natural forest. As a result, shrubs and trees that are resistant to grazing have the highest share.\n\nClimbers were present in some forest patches, particularly in highland and mid-altitude forest patches, which adds to the beauty of the forest in mountainous areas. This is strongly connected to human and grazing issues, which are especially severe in lowland places with few climbing plants. Life forms, notably herbs, and climbers, are highly susceptible to disturbances. According to Mahdavi et al. (2013) and Shary et al. (2020), habitat instability is the fundamental factor that affects the proportion of life forms.\n\nWoody species structure\n\nLowland and mid-altitude forest patches exhibit inverted-J structures, whereas highland forests have J-shaped structures (Figure 5A & B).\n\nHowever, there are extremely few seedlings in all forests and agroclimatic zone, implying that no trees have less than 5 cm DBH. The mid-latitude forest has an inverted J-shape structure based on DBH class distributions, indicating that higher forest tree populations were found in lower diameter classes, however, this does not imply that the forest is a healthy community. According to Senbeta et al. (2014), forest patches with an inverted-J shape indicate good natural regeneration and a healthy community. There were few trees having seedlings less than 5 cm in diameter in all forest patches, indicating a lack of spontaneous regeneration (Figure 5C). In highland agroclimatic zone with intermediate anthropogenic disturbances (Table 4), the vegetation pattern has a J-shape, indicating a healthy forest ecosystem.\n\nHowever, there is still a natural regeneration issue, and seedlings are unable to reach the sapling and tree phases. This is due to human intervention and grazing encroachment. Meragiaw et al. (2018) endorse this theory, stating that the lowest percentage of species found in the lowest and highest classes could be due to the overgrazing of young plants and selective extraction of young trees. Several scholars (Atomsa and Dibbisa, 2019; Mewded, Negash, and Awas, 2019; Dibaba et al., 2020; Tadese et al., 2021) argued that in some parts of Ethiopia woody species population structure was follow either or a combination of inverted J-shape, a J-shape, a bell-shape, and an irregular shaped pattern which is an indicator of high anthropogenic disturbances combined with environmental factors that affect natural forest community balance. As a result, such pattern variability is an indicator for immediate intervention steps to ensure the survival and sustainability of forests that are experiencing poor regeneration.\n\nWoody species dominance\n\nAccording to the important value index, Schefflera abyssinica dominated highland forest patches, followed by Dombeya torrida and Albiza gumifera, whereas Syzygium guineense was the least dominant species, despite being an ecologically and economically important indigenous woody species. Because of its large basal area, Schefflera abyssinica was the dominating species, but it had a low stem density (Figure 6A). Whereas Mimusops kummel was the dominant species in mid-altitude forest patches due to its largest basal area, Albiza gumifera, and Prunus africana were the prevalent species in highland and mid-altitude forest patches. Despite being a keystone species, Teclea nobilis had the lowest IVI in mid-altitude forest patches (Figure 6B). In lowland forest patches, Oxytenanthera abyssinica and Ficus sycomorus were the most dominant species, followed by Combretum nigricans. Ziziphus mumucronta was the least prevalent species, despite being a wild edible fruit species that plays an important ecological role in lowland forest patches (Figure 6C).\n\nBecause of their economic relevance in timber and non-timber products, the species with the least dominance have suffered degradation due to over-utilization. Several studies (Kidane, Stahlmann, and Beierkuhnlein, 2012; Sales de Melo et al., 2019; Mucheye et al., 2020) contended that species with high economic value had lower important value indexes, indicating degradation. As a result, they require unique conservation priorities that promote natural regeneration as in-situ conservation and collection in arboretums as ex-situ conservation.\n\nHeight, diameter, and stem density differed significantly (p 0.03) within and within agroclimatic zones. Mid-altitude forest patches had the highest stem density (2578 trees per ha), followed by highland (1351 trees per ha) and lowland (1268 trees per ha). Highland forest patches have the highest height (25.14m) and diameter (40.41 cm), followed by mid-altitude (19.5m height and 26.8cm diameter) and lowland forest patches (16.03m height and 25.9 cm diameter) (Table 3).\n\nHeight, diameter, and stem density differed significantly (p 0.03) across slope gradients; however, changes in aspect and elevation gradients did not contribute to a significant variance (p 0.05) within forest patches. The moderate slope had the highest stem density (2260 trees per hectare), whereas the medium slope (2124 trees per ha) and steep slope (1468 trees per ha) had the lowest (Table 1).\n\nHighland forest patches exceed mid-altitude and lowland forest patches in diameter and height growth. Better climatic conditions and anthropogenic perturbations may be substantially connected with growth performance and growth rate. This notion is comparable to that of Toledo et al. (2011), who show that climatic and edaphic conditions fluctuate with place and time, influencing growth performance appropriately. Mid-altitude forest patches, on the other hand, outperform highland and lowland forest patches in terms of stem density. Lowland forests had a poor growth performance in any case. This could be due to human and grazing effects because there was extensive prohibited cutting and overgrazing in lowland forest patches, resulting in poor growth performance (Table 4).\n\nFlat slopes have the highest stem density in an open natural forest, followed by medium and steep slopes. Flat slopes have higher soil fertility and ideal climatic conditions, which subsidize tree micro-sites for better plant growth and accumulation (Gemedo et al., 2014; Field, 2014; Senbeta et al., 2014). Furthermore, Afromontane forests have a high density of woody plants that vary greatly between places (Senbeta et al., 2014).\n\nAt a significant level of p 0.05, forest patches and human impacts in all agroecological clusters incur a similar degree of effect. However, there was a substantial difference in grazing impact and vegetation cover-abundance within and between agroclimatic zones (p 0.005) (Table 4). This suggests that all forest sections around the globe are being subjected to extensive tree cutting by illegal cutters and grazing encroachment. As a result, lowland forests have the biggest grazing issue, followed by mid-altitude and highland forests, and it has a substantial impact on the cover-abundance of lowland forest patches. Mid-altitude forest patches had the most abundant but interrupted vegetation cover (5 = 50-75% cover), followed by highland forests with abundant cover (4 = 26-50%) and lowland forests with less than 3 = 6-25 rare vegetation cover (Table 4). Intensive grazing and human encroachment threaten lowland forest patches. This is due to the lowland areas’ animal-based farming practices. That is why vegetation cover-abundance is greater in mid-altitude and highland forest patches than in lowland forest patches. This could be attributed to environmental compatibility as well as human and grazing intensity. Human effect was substantial in all forests and agroclimatic zone, whereas grazing impact is lowland > mid-altitude > highland.\n\nThus, anthropogenic disturbances with the interaction of climate variances are the primary determinants of vegetation cover-abundance. This argument is similar to Alemayehu (2007) and Cardelús et al. (2019) claim that anthropogenic disturbances are common in all forest habitats and have a detrimental impact on variety, growth performance, and vegetation cover-abundance. Furthermore, most evaluated forest patches encounter similar levels of disturbance, and the forest population is significantly threatened by habitat fragmentation, lack of regeneration, and high disturbances (Chetcuti, Kunin, and Bullock, 2022; Pinho et al., 2022).\n\nExternally, almost all study forest patches appear intact; yet, they are open on the interior, and only large trees shield the canopy, as observed in vegetation structure (Figure 5). There is little regeneration and very little conservation. All of this is the result of extensive human encroachment and overgrazing. This result is consistent with the findings from Teketay (1992), regarding the subjectivity of most Ethiopian natural forests to strong pressure and, as a result, the risk of local extinction. In addition, White and Hood (2004) contend that grazing has an impact on natural regeneration, life in transition, and optimal productivity. As a result, grazing and human encroachment have altered growing habitats, influencing vegetation dynamics (Zhang et al., 2013; Field, 2014; Girma, Chuyong, and Mamo, 2018; Cardelús et al., 2019).\n\n\nConclusions\n\nSlope, agroclimatic zone, and forest patches have a substantial influence on the vegetation features of the evaluated forest patches, with altitude and aspect having a minor influence. Forest patches in close spatial proximity have similar species diversity and composition, whereas those with large-scale altitude differences show significant disparities. Transitional forest patches that share some species between the extremes, on the other hand, show small parallels in species composition and diversity. The forests in the study area are open natural forests that are subject to anthropogenic habitat disturbances, resulting in a higher proportion of trees and shrubs. This emphasizes the importance of disturbances and habitat instability in the spread of plant life forms.\n\nSatellite species were detected throughout all agroclimatic zone and elevation gradients, with low abundance and inconsistent distribution. This implies that native floristic species have low abundance and distribution, most likely because of deforestation and forest degradation, both of which act as indications of local species extinction. The woody species population structure in the study forest patches follows various patterns, including a combination of inverted J-shaped, J-shaped, bell-shaped, and irregular-shaped patterns, indicating high anthropogenic disturbances combined with environmental factors that affect the balance of natural forest patches, like other parts of Ethiopia.\n\nHuman activities and grazing are common in all forest patches and have a negative impact on floristic diversity, composition, growth performance, vegetation distribution, structure, and cover abundances, as well as long-term effects on habitat fragmentation. Most forest patches in various agroclimatic zones have been impacted by humans and livestock, resulting in reduced natural regeneration and high levels of deforestation and forest degradation. While the forests appear green and lush from the outside, most species have not naturally regenerated, and the canopy is dominated by massive, elderly trees that are becoming increasingly vulnerable to illegal cutting. This makes the forest patches less valuable and more vulnerable to deterioration. Woody species that are less prominent in the study forest patches have suffered deterioration because of overutilization for their economic worth in timber and non-timber products.\n\nAs a result of the observed pattern of vegetation structure, composition, and distribution variability, as well as the risk of local extinction, urgent intervention is required to ensure the survival and sustainability of sensitive woody species suffering from poor regeneration status. As a result, extraordinary conservation measures are required, with the active participation of forest researchers, development agents, and political leaders, to safeguard the few remaining natural forests. If the encroachment is not addressed, critical biological and socioeconomic forest patches will be lost soon. Conservation actions aiming at conserving vegetation and wildlife habitats should be prioritized.\n\n\nEthics approval and consent\n\nAnimals, humans, and other plant propagules were not used in this research. Thus, it is not relevant to this work.", "appendix": "Data availability\n\nMendeley Data: The datasets generated during and/or analyzed during the current study are available at Mekonen (2024), “Floristic diversity of natural forest patches across agroclimatic zones in Northwestern Ethiopian”, Mendeley Data, V2, https://www.doi.org/10.17632/g889p49pb9.3 (Mekonen et al., 2024)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgments\n\nFirst, we would like to thank Injibara University for its arrangement for transport facilities and measurement materials. Lastly, I would like to thank the university colleges and supporting staff who helped complete this work.\n\n\nReferences\n\nAide TM, et al.: Woody vegetation dynamics in the tropical and subtropical Andes from 2001 to 2014: Satellite image interpretation and expert validation. Glob. Chang. Biol. 2019; 25(6): 2112–2126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlemayehu W: Ethiopian Church Forests: Opportunities and Challenges For Restoration. Wageningen Univeristy; 2007.\n\nAngessa AT, et al.: Woody plant diversity, composition and structure in relation to environmental variables and land-cover types in Lake Wanchi watershed, central highlands of Ethiopia. Afr. J. Ecol. 2020 (April 2019); pp. 1–12. Publisher Full Text\n\nAtomsa D, Dibbisa D: Floristic composition and vegetation structure of Ades forest, Oromia regional state, West Hararghe zone, Ethiopia. Trop. Plant Res. 2019; 6(December 2018): 139–147. Publisher Full Text\n\nAwi ZOne Agricultural office: Awi Zone Agricultural Offices Annual Report. Injibara; 2021.\n\nBekele-Tesema A: Useful trees and shrubs for Ethiopia: identification, propagation and management in 17 agro-ecological zones. 2nd edn, RELMA in ICRAF Project. 2nd ed.Nairobi: RELMA in ICRAF Project; 2007.\n\nBelay TA, Totland Ø, Moe SR: Woody vegetation dynamics in the rangelands of lower Omo region, southwestern Ethiopia. J. Arid Environ. 2013; 89: 94–102. Publisher Full Text\n\nCardelús CL, et al.: Human disturbance impacts the integrity of sacred church forests, Ethiopia. PLoS One. 2019; 14(3): 1–14. Publisher Full Text\n\nCaroline M, et al.: Can small - scale altitudinal gradients predict spatial and temporal patterns in tropical forests? J. For. Res. 2020. [Preprint]. Publisher Full Text\n\nChetcuti J, Kunin WE, Bullock JM: ‘Species’ movement influence responses to habitat fragmentation. 2022; pp. 1–14. (August). Publisher Full Text\n\nCoomes OT, et al.: Environmental and market determinants of economic orientation among rain forest communities: Evidence from a large-scale survey in western Amazonia. Ecol. Econ. 2016; 129: 260–271. Publisher Full Text\n\nCormack R, Jeffers JNR, Colinvaux PA: Vegetation Dynamics. Dunnet GM, Gimingham CH, editors. 1st edn.London: John Miles; 1979. Publisher Full Text\n\nDang Hoi N, Trung Dung N: Seasonal Dynamics of Tropical Forest Vegetation in Ngoc Linh Nature Reserve, Vietnam Based on UAV Data. For. Soc. 2021; 5(November): 376–389. Publisher Full Text\n\nDella Rocca F, et al.: Effects of natural and seminatural elements on the composition and dispersion of carabid beetles inhabiting an agroecosystem in Northern Italy. Ecol. Evol. 2021; 11(15): 10526–10537. Publisher Full Text\n\nDibaba A, et al.: Woody Species Diversity, Vegetation Structure, and Regeneration Status of the Moist Afromontane Forest of Agama in Southwestern Ethiopia. Int. J. Ecol. 2020; 2020.\n\nDwyer C, et al.: Patterns of variation in plant diversity vary over different spatial levels in seasonal coastal wetlands.2022; pp. 1–16. (May). Publisher Full Text\n\nFAO: National forest inventory field manual tamplate. Rome: 2004; 94.\n\nFAO: Guidelines for Soil Description. FAO. 4th edn.Rome (Italy): FAO; 2006. Publisher Full Text\n\nField CB: Linking vegetation patterns to environmental gradients and human impacts in a mediterranean-type island ecosystem. Landsc. Ecol. 2014; 1571–1585. Publisher Full Text\n\nFranklin E v d M: Vegetation ecology. Second, Choice Reviews Online. Second ed.Arizona State University, USA: A John Wiley & Sons, Ltd., Publication; 2013. Publisher Full Text\n\nGemedo D, Maass BL, Isselstein J: Relationships between vegetation composition and environmental variables in the borana rangelands, southern Oromia, Ethiopia. Ethiop. J. Sci. 2014; 37(1): 1–12.\n\nGenet A: Indigenous institutions and their role in socioeconomic affairs management: Evidence from indigenous institutions of the Awi people in Ethiopia. Int. Soc. Sci. J. 2022; 72(243): 225–241. Publisher Full Text\n\nGirma Z, Chuyong G, Mamo Y: Impact of Livestock Encroachments and Tree Removal on Populations of Mountain Nyala and Menelik’s Bushbuck in Arsi Mountains National Park, Ethiopia. Int. J. Ecol. 2018; 2018. Publisher Full Text\n\nGorfu D, Ahmed E: Crops and Agro-ecological Zones of Ethiopia.2011; 47. Reference Source\n\nGotelli NJ, Anne C: Measuring and Estimating Species Richness, Species Diversity, and Biotic Similarity from Sampling Data Encyclopedia of Biodiversity. 2nd ed.Waltham: Elsevier Ltd.; 2013. Publisher Full Text\n\nGray JS, Ugland KI, Lambshead J: On species accumulation and species-area curves. Glob. Ecol. Biogeogr. 2004; 13(6): 567–568. Publisher Full Text\n\nHawk GM, Long JN, Franklin JF: Relations Between Vegetation and Environment.1977\n\nHull JC, Neufeld HS, Gilliam FS: Plant ecology. Encyclopedia of Ecology. 1st ed.Heidelberg: Springer Berlin Heidelberg; 2018. Publisher Full Text\n\nJyothi PV, Sureshkumar S: Patterns of Vegetation Dynamics across Mild Disturbance Gradient in a Freshwater Wetland System in Southern India. Wetlands. 2018; 38(4): 807–817. Publisher Full Text\n\nKent: Vegetation Description and Analysis: A Practical Approach. Second, The Geographical Journal. Second ed.Atrium, Southern Gate, Chichester, West Sussex: A John Wiley & Sons, Ltd., Publication; 2012. Publisher Full Text\n\nKidane Y, Stahlmann R, Beierkuhnlein C: Vegetation dynamics, and land use and land cover change in the Bale Mountains, Ethiopia. Environ. Monit. Assess. 2012; 184: 7473–7489. Publisher Full Text\n\nLemessa D, et al.: Woody Species Composition in Relation to Spatial and Environmental Gradients in Acacia-Commiphora Vegetation Ecosystem of Ethiopia. Int. J. Nat. Resour. Ecol. Manage. 2017; 2(3): 53–59. Publisher Full Text\n\nMahdavi P, Akhani H, Van der Maarel E: Species Diversity and Life-Form Patterns in Steppe Vegetation along a 3000 m Altitudinal Gradient in the Alborz Mountains, Iran. Folia Geobot. 2013; 48(1): 7–22. Publisher Full Text\n\nMartín-Devasa R, et al.: Species range size shapes distance-decay in community similarity. Divers. Distrib. 2022; 1348–1357. (April). Publisher Full Text\n\nMekonen M, Derso Y, Asres M, et al.: Floristic diversity of natural forest patches across agroclimatic zones in Northwestern Ethiopian. Dataset. Mendely Data. 2024. Publisher Full Text\n\nMeragiaw M, et al.: Woody species composition and diversity of riparian vegetation along the Walga River, Southwestern Ethiopia. PLoS One. 2018; 13(10): 1–18. Publisher Full Text\n\nMewded B, Negash M, Awas T: Woody species composition, structure and environmental determinants in a moist evergreen Afromontane forest, southern. J. For. Res. 2019. [Preprint]. Publisher Full Text\n\nMucheye T, et al.: Long - term dynamics of woodland vegetation with response of climate variability in the lowlands of north western part. Environ. Dev. Sustain. 2020. [Preprint]. (0123456789). Publisher Full Text\n\nMusau J, et al.: Spatio-temporal vegetation dynamics and relationship with climate over East Africa. Hydrol. Earth Syst. Sci. Discuss. 2016; pp. 1–30. (October). Publisher Full Text\n\nNaidu MT, Kumar OA: Tree diversity, stand structure, and community composition of tropical forests in Eastern Ghats of Andhra Pradesh, India. J. Asia-Pac. Biodivers. 2016; 9(3): 328–334. Publisher Full Text\n\nNzabarinda V, et al.: Assessment and evaluation of the response of vegetation dynamics to climate variability in Africa. Sustainability (Switzerland). 2021; 13(3): 1–22. Publisher Full Text\n\nPease BS, et al.: What drives spatially varying ecological relationships in a wide-ranging species? Divers. Distrib. 2022; pp. 1752–1768. (June). Publisher Full Text\n\nPinho BX, et al.: Cross-scale drivers of woody plant species commonness and rarity in the Brazilian drylands. Divers. Distrib. 2022; 28(7): 1497–1511. Publisher Full Text\n\nRosindell J, Cornell SJ: Species - area curves, neutral models, and long-distance dispersal. Ecology. 2009; 90(7): 1743–1750. Publisher Full Text\n\nSales de Melo CLSM, et al.: Dynamics of dry tropical forest after three decades of vegetation suppression. Floresta e Ambiente. 2019; 26(3): 1–12. Publisher Full Text\n\nScheiner SM: Six types of species-area curves. J. Glob. Ecol. Biogeogr. 2003; 12: 441–447.\n\nSenbeta F, et al.: Plant Diversity, Vegetation Structure and Relationship between Plant Communities and Environmental Variables in the Afromontane Forests of Ethiopia. Sci. J. Ethiop. 2014; 37(2): 113–130.\n\nShary PA, et al.: Impact of Environmental Factors on the Life-Form Diversity of Grassland Vegetation in the Southern Forest–Steppe. Russ. J. Ecol. 2020; 51(1): 11–19. Publisher Full Text\n\nSoromessa T, Teketay D, Demissew S: Ecological study of the vegetation in Gamo Gofa zone, southern Ethiopia. Trop. Ecol. 2004; 45(2): 209–221.\n\nTadese S, et al.: Woody Species Composition, Vegetation Structure, and Regeneration Status of Majang Forest Biosphere Reserves in Southwestern Ethiopia. Int. J. For. Res. 2021; 2021.\n\nTeketay D: Human Impact on a Natural Montane Forest in Southeastern Ethiopia. Mt. Res. Dev. 1992; 12(4): 393–400.\n\nToledo M, et al.: Climate is a stronger driver of tree and forest growth rates than soil and disturbance. J. Ecol. 2011; 99(1): 254–264. Publisher Full Text\n\nTuxen R: Handbook of Vegetation dynamics. Knapp R, editor. 1st edn.HAGUE: Dr. W. JUNK h.v.-PUBLISHER; 1974.\n\nWhite DA, Hood CS: Vegetation patterns and environmental gradients in tropical dry forests of the northern Yucatan Peninsula. J. Veg. Sci. 2004; 15(2): 151–160. Publisher Full Text\n\nWondie M, Mekuria W: Planting of acacia decurrens and dynamics of land cover change in fagita lekoma district in the Northwestern Highlands of Ethiopia. Mt. Res. Dev. 2018; 38(3): 230–239. Publisher Full Text\n\nZhang J, Xu B, Li M: Vegetation Patterns and Species Diversity Along Elevational and Disturbance Gradients in the Baihua Mountain Reserve, Beijing, China. Mt. Res. Dev. 2013; 33(2): 170–178." }	[ { "id": "313507", "date": "03 Sep 2024", "name": "Biljana Lubarda", "expertise": [ "Reviewer Expertise plant distribution", "plant diversity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is clearly and meaningfully written. The paper analyzes floristic diversity, floristic species similarity, floristic species distribution, woody species structure, woody species dominance, woody species growth performance, as well as human and grazing impact on natural forest. It is interesting that the authors came to the conclusion that \"Slope, agroclimatic zone, and forest patches have a substantial influence on the vegetation features of the evaluated forest patches, with altitude and aspect having a minor influence\". I ask a question: aren't agroclimatic zones related to altitude? If this is not the case in the researched area, you can ignore my comment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "313504", "date": "19 Sep 2024", "name": "Belachew Bogale Worku", "expertise": [ "Reviewer Expertise Forestry", "Green Development and Carbon Management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is someone good, but have the following specific comments to be addressed. #1 Abstract:\nIn the abstract, the author concluded that “according to the study’s findings, the state of forest resources varies considerably across different environmental variations. Despite the impression of entire forest patches from the outside, the interiors are open, with only huge and mature trees covering the canopy.” Here the word “huge” leads paradoxically to the context of “the interiors are open.”. Thus, it needs reshaping of the sentences to be agreed with “the interiors are open.\n#2 Keywords:\nKeywords are usually written in alphabetical order; however, the author states in disorderly. Hence, ordering it in accordance with “environmental factors, floristic diversity, forest patches, vegetation distribution, vegetation structure” is required.\n#3 Introduction:\nThe introductory section lacks topic, like “Introduction”. The first two sentences in paragraph one have shown some duplicated terms that have to be refined. Look at these sentences “Forest vegetation is an important natural resource with global ecological, economic, and social implications. Forest vegetation is an important component of the biogeochemical cycle and aids in the protection of the earth from natural and anthropogenic risks, particularly in moderating climate change.” In this case, the term “Forest vegetation is important” becomes a sentence beginning phrases for both sentence one and two. This indicates sentence one and two can be summarized and become one sentence. Paragraph 2: Old references is cited in the first sentence of paragraph two. E.g., Cormack et al., 1979.” Similarly, old references is used in result section (Hawk et al., 1977). Look at these sentences “The principal sources of temporal and spatial changes in vegetation are natural plant development processes and disturbances (Hull et al., 2018). Anthropogenic and natural disturbances have a significant impact on forest ecosystem vegetation, with time and place variations. Time is considered to be a key factor in vegetation dynamics (Cormack et al., 1979). Rainfall, temperature, wind, light, humidity, and other climatic variables change throughout time and influence ecological variables that affect plant ecology and evolution.” They have the same meaning; need summarizing.\n#4 Study area description\nThe study area has an altitudinal range of 600 to 3500 m. a. sl., 1750 mean annual rainfall, and temperatures ranging from 170°C to 27°C. In this sentence, the authors required to correct temperature value (170°C by 17°C). Correct the sentence “Agroecologically, most of the Awi administrative Zone is highland (17%), and lowland (11%)” into “Agroecologically, most of the Awi administrative Zone is mid-altitude (72%), followed by highland (17%), and lowland (11%).”\n#5 Sampling Techniques and Data Collection\nHighland (2200-3600 m.ab.sl.), Mid-altitude (1300-2200 m.ab.sl.), and Lowland (500-1300 m.ab.sl.) were the primary Awi Zone classifications (Bekele, 2007; Gorfu and Ahmed, 2011). In this sentence, the authors used abbreviation, such as m.ab.sl, but it has to be written in the manner of m.a.s.l. The main plot was 20 m by 20 m for trees, with two 10 m by 10 m sapling subplots, five 5 m by 5 m seedling and regeneration subplots, and five 1 m by 1 m herbaceous species subplots within the main plots. In this sentence, authors used two 10 by 10 m sapling plots in the main plot, which do not have any difference from the main plot, 20 by 20 m. What base line you use to make the sapling plots double 10 by 10 m? Any references is required to convince the scientific community. The author stated that “Following standard inventory procedures, tree growth parameters (height and DBH) were measured with a hypsometer and diameter tape, respectively, and the life form of a species was clustered as trees (DBH > 2.5 cm and height of >2.5 m), saplings (DBH 2.5 cm and height of 1m h 2 m), and seedlings and herbs (DBH 2.5 cm and height of 1 m) at each plot (FAO, 2004; Kent, 2012).” In this sentence, life forms of plant species is classified as trees, saplings, seedlings and herbs, which is totally wrong because classifying of plants into trees, saplings, and seedlings indicates developmental stage rather than life forms. Life forms mainly stated as trees, shrubs, herbs, etc. The other thing which needs correction is DBH of herbaceous species. As far as my knowledge, it is not possible to measure DBH for herbaceous species. Therefore, the sentence needs some correction. See Table 2 for clarity.\n#6 Data Analysis\nThe author stated that “Forest species were identified using NDA (Natural Database Africa) software Ver. 2.0 and the Flora of Ethiopia and Eritrea book (Bekele, 2007), vegetation descriptions, and environmental patterns were analyzed using Kent (2012), Zhang et al. (2013), Lemessa et al. (2017) methods.” The problem in this statement is citation for “the Flora of Ethiopia and Eritrea book (Bekele, 2007). Flora of Ethiopia and Eritrea books are written by other authors and Bekele did not involved in this work but rather had his own book, such as “useful trees and shrubs in Ethiopia.” Authors should take care of using such mismatched authors to the published works, which leads not abiding for scientific ethical consideration of paper writing. Besides, species identification using NDA (natural Database Africa) is not recommended since NDA has many problems, including mismatching of species generic names and scientific names.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-811
https://f1000research.com/articles/13-810/v1	17 Jul 24	{ "type": "Research Article", "title": "Absent in Melanoma 2 Gene Associated Periodontitis and Coronary Heart Disease", "authors": [ "Zina Ali Daily", "Batool Hassan Al-Ghurabi", "Batool Hassan Al-Ghurabi" ], "abstract": "Aims To study the association between AIM2 gene polymorphisms and the tendency for periodontal infection and coronary heart disease, and to determine whether males or females are more susceptible to these diseases. Additionally, we examined its association with the features of periodontal disease.\n\nMethods 140 patients were enrolled in this study, and those who took part were divided into four groups as follows: healthy (c), periodontal disease (P), coronary heart disease with intact periodontium (AS-C), and coronary heart disease with periodontal disease (AS-P). Information on entrants, including age, sex, body mass index, and indicators of periodontal disease severity, was documented. Blood samples were collected, and AIM2 gene polymorphisms were evaluated by polymerase chain reaction test, gel phase, and sequences.\n\nResults Genetic analysis of AIM2 G/T (rs2793845) revealed a high frequency of the (T) allele and (GT and TT) genotypes that were detected in the periodontal disease and coronary heart disease groups in males. The Hardy-Weinberg equilibrium of alleles and genotypes did not differ significantly between the study groups. Gene polymorphisms were also significantly correlated with indicators of periodontal disease severity.\n\nConclusion High frequenting of (T) alleles and (GT, TT) genotypes in AIM2 single nucleotide polymorphisms (SNP) were associated with an increased tendency to develop periodontal disease and coronary heart disease. It can be supposed that it has a causative function in the pathophysiology of both disorders, and the validity of SNP as a potential genomic factor for the risk of both disorders in Iraqi males.", "keywords": [ "periodontal disease", "coronary heart disease", "gene polymorphism", "AIM2 gene." ], "content": "Introduction\n\nThe tissue connection and support teeth in position are demolished by an infectious inflammatory illness known as periodontitis. By transitioning from a symbiotic to a dysbiotic condition, pathobionts also harm the tissue.1,2 Immune responses are mostly controlled by genotypes, and dysbiotic flora also influences the response. The distribution and severity of diseases are influenced by genetic factors, which also have a substantial impact on the immune response to bacterial infections.3 In response to an increase in the levels of lipids and low-density lipoprotein (LDL) in the blood, the immune system launches, propagates, and triggers atheroma throughout the circulatory system, resulting in atherosclerosis (AS), an inflammatory disease. Atherosclerosis is one sign of coronary heart disease (CHD).4,5 An assembly of several proteins, known as the AIM2 inflammasome, occurs in response to microbiome or sterile allusions. During apoptosis, inflammatory mediators are released into cells as a result of the active form of caspase-1. Moreover, it is linked to osteoclast stimulation, atheroma formation, and progressive periodontal ligament injury.6–8 Numerous studies have documented a correlation between genetic variation in the inflammasome constituent genomes and heightened tendency toward periodontal disease or CHD among diverse ethnic groups.9,10 Genetic illnesses, such as autoimmune sickness, dermatitis, and periodontal disease, have also been reported to be associated with variants of the AIM2 genetic factor, known as DNA, which consists of two strands of sensors.11 To date, there is no evidence from any research linking a variant of the AIM2 gene to periodontal disease risk in people with or without CHD. This sheds insight into the current investigation, which aims to determine the relationship between AIM2 gene polymorphisms and periodontal disease propensity in both people with and without CHD, and to detect whether males or females are more susceptible to these diseases. We also evaluated the correlation between AIM2 single nucleotide polymorphisms and indicators of periodontal disease severity.\n\n\nMethods\n\nCase-control study design from November 2022 to May 2023, the inquiry occurred in numerous centers in Baghdad City. The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the University of Baghdad, College of Dentistry (Ref. 652, 13/09/2022, Project # 652622) that gave their stamp of approval to the study’s moral foundations. All the human research protocols complied with the ethical standards laid out in the Helsinki Statement and its later modifications. Each participant signed an informed written consent form after being provided detailed information about the study and its purposes. The present study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) in terms of design of the study and results reporting.\n\nThis investigation evaluated 140 entrants, including males and females, aged range (40-70) years, all with an ordinary range of body mass index (BMI) who were recruited in this study according to the inclusion and exclusion criteria. The entrants were diagnosed according to the 2017 classification of periodontal diseases and conditions with severity of cases in periodontal disease groups.12,13 The indicators of periodontal disease were clinically assessed using a UNC-15 probe for each tooth, including CAL and PPD, which were measured to the nearest millimeter, the whole-mouth plaque index (PLI),14 and whole-mouth BOP.15 The diagnosis was made by a cardiologist specialist for CHD through the coronary arterial catheter technique, presenting current atheroma in excess of 75%.16–19\n\nInclusion criteria included individuals (a) through an acceptance to participate in the investigation, (b) in excellent general wellness, other than a diagnosis of atherosclerosis from a heart catheter technique, and without any other systemic illnesses. Elimination standards involved individuals (a) with physical disorders, (b) undergoing periodontal therapy within the previous period of six months, (c) were smoking behavior, (d) had reserved inflammation inhibitor medicine in the previous period of three months; and (e) were either breastfeeding or pregnant when the research was conducted. Following the recruitment of research entrants, each participant signed a declaration of consent. After that, every entrant’s blood sample was taken, clinical evaluations were thoroughly documented, and demographic details were noted.\n\nGathering and examination of blood samples in less than 30 s, 2 mL of venous blood was pulled, placed in an ethylenediaminetetraacetic acid (EDTA) tube, and kept at -40°C for the (AIM2) genome using PCR-sequencing techniques. Genomic DNA was extracted from the blood samples using the ReliaPrep Blood gDNA Miniprep System (Promega, USA) DNA purification kit, and the isolated DNA was assessed using a Quantus Fluorometer. To create a functional primer solution, the lyophilized form was submerged in nuclease-free water (NFW). The appropriate temperature for primer annealing was also determined. The DNA template was multiplied using identical forward and reverse primer pairs at the annealing DNA template. PCR enhancements were then performed. Following PCR augmentation, multiplication was demonstrated using electrophoresis with agarose gel (OWL, Thermo, USA) combined with 10 mg/mL ethidium bromide stain (EBS) (Promega, USA), as shown in Figure 1. The PCR outcomes were analyzed by Sanger sequencing using a Macrogen ABI3730XL Automated Genomic Sequencer (Korea).\n\nAn investigation of the same examiner’s validity was necessary to determine the dependability of this study. After assessing five patients with periodontal disease, two calibrating meetings for indicators of periodontal disease severity were obtained within an hour. For PPD and CAL, the intraclass coefficient was 0.96, while BOP and PLI had kappa coefficient assessed 0.85 and 0.94. Consequently, the dependability level of the investigation was adequate.\n\nThe main genetic marker of AIM2 in the research groups was identified on the basis of the findings of the pilot study. With an estimated power of 80% and an alpha likelihood ratio of 0.05, an overall number of 140 entrants (35 individuals for each group) were determined for the sample size using Odds/Ratios and https://epitools.ausvet.com.au/casecontrolss.\n\nGraph Pad software Inc. La Jolla, CA’s Prism version 9.0 was used to do the statistical evaluation.20 The one-way analysis of variance and the average and deviations for every of the categories’ separate variable. The Odds/Ratio method was used to determine frequencies of allele and genotype. Pearson correlation coefficient of gene polymorphism with indicators of periodontal disease severity.\n\n\nResults\n\nA total of 1370 people examined to participate in the current investigation, whereas 1230 individuals declined based on the grounds for exclusion, as illustrated in (Figure 2). The average value was computed considering the demographic factors of the 140 individuals in the four groups (healthy (c), periodontal disease (P), coronary heart disease with intact periodontium (AS-C), and coronary heart disease with periodontal disease (AS-P)). The sex distribution of all groups was 80% males and 20% females. This study examined the body mass index (BMI) of individuals with a weight of 24 kg/m2. Variations in age, sex, and body mass index across the groups were not statistically significant (p ≤ 0.05) (Table 1). Moreover, the present outcomes determined a significant increase in the average values of indicators of periodontal disease severity in the periodontitis patient groups. Sequencing of the AIM2 SNP using the Sanger method in the four study groups is shown in Figure 3. This SNP, rs2793845, results from the replacement of guanine (G) with thymine (T) in the intron region of one chromosome.\n\n* significant at p ≤ 0.05, PLI, plaque index; BOP, bleeding on probing; PPD, probing pocket depth; CAL, clinical attachment loss.\n\nA solitary “T” top point to the presence of a T homozygous for allele. A solitary “G” top point to the presence of a G homozygous for allele. The existence of the “G” and “T” points suggests the existence of the G/T heterozygous allele.\n\nFurthermore, the present study was identified TT genotype of AIM2 G/T (rs2793845) was a high odd/ratio (OR=14.9 (3.990 to 45.98), P-value=0.0) in P group, (OR=16.43 (0.5094 to 0.4878), P-value = 0.0013) in AS-C group and (OR=16.45 (0.05094 to 0.4878), P-value=0.0013) in AS-P group as compared to control healthy group, whereas genotype GT was a high odd/ratio (OR=22.44 (0.009776 to 0.7495), P-value=0.0227) in P group, (OR=9.12 (0.007127 to 0.6108), P-value=0.0056) in AS-C group and (OR=6.93 (0.004114 to 0.3088), P-value=0.0002) in AS-P group than control healthy group, (p-value<0.000), as described in Table 2. Data analysis of alleles frequencies in patients and controls groups are summarized in Table 3, the T allele of the AIM2 G/T (rs2793845) gene showed greater frequenting in the P group was (0.85), AS-C group was (0.72), and AS-P group was (0.8) compared to the G allele, and it is slightly higher than the allele frequency in Asians was 0.65 and the global population was 0.86 from the Genome Aggregation Database (gnom AD). In addition, the frequencies of alleles and genotypes in the AIM2 gene polymorphism did not show any substantial variation relative to the Hardy-Weinberg equilibrium (HWE) in any group of participants. The correlation of AIM2 with indicators of periodontal disease severity in the patient groups is shown in Table 4. There was a significant correlation between AIM2 G/T (rs2793845) and BOP (r=0.444, p=0.013; r=0.463, p=0.030), PPD (r=0.364, p=0.048; r=0.399, p=0.029), and CAL (r=0.504, p=0.005; r=0.536, p= 0.004) in the P group and AS-P group, respectively.\n\n* significant at p ≤ 0.05,\n\n** significant at p ≤ 0.01, Chi χ2 = Chi-square, RR = Relative risk, OR = Odds ratio, HWE χ2 = Hardy-Weinberg Equilibrium chi-square.\n\n* significant p value ≤ 0.05.\n\n* Significant at p < 0.05; r, Pearson’s coefficient correlation; PLI, Plaque indicator; BOP, bleeding on Probing; PPD, Probing Pocket depth; CAL, clinical attachment loss.\n\n\nDiscussion\n\nThe current investigation identified a correlation between genomic variations related to inflammation in individuals and increased vulnerability to developing periodontitis, both with and without coronary heart disease. This was achieved by analyzing certain genotypes that played a role in the progress of both conditions. Additionally, indicators of periodontal disease severity and the AIM2 gene showed a significant association. The results revealed that males were more susceptible to developing these diseases in all groups at a ratio of 80 males and 20 females. In the AIM2 (rs2793845) gene, the P, AS-C, and AS-P groups had noticeably greater frequencies of T allele and distribution (GT, TT) genotypes in blood samples than the control group. An analysis of the AIM2 gene’s single nucleotide polymorphism linked the AIM2 gene’s downstream functional area to the occurrence of periodontitis and coronary heart disease through abnormal gene expression and activation of AIM2 proteins and host molecules. This results in the degradation of the periodontium, impaired functioning of endothelial cells, and production of plaques that contribute to the development of atherosclerosis. This SNP influences the process of inflammation through changes in the shift-regulating activity. Therefore, the presence of these abnormal genetic variations in AIM2 was linked to a higher likelihood of developing periodontitis, both with and without coronary heart disease. Marchesan et al. found that the frequency of a specific gene variant (allele) and the distribution of different genetic combinations (genotypes) of the AIM2 gene (specifically SNP rs1057028) may have a substantial impact on the development of periodontal diseases.21 In contrast, Figueira et al. observed that individuals with a specific genetic variation (SNP) in the AIM2 gene (rs1103577) had a considerably reduced chance of developing pulmonary tuberculosis (PTB) compared to healthy individuals.22 In polymorphisms of the AIM2 gene, the frequency of the T allele appears to be slightly higher than that in Asians and in the global population. In addition, the association of T alleles of the AIM2 SNP with the probability of periodontal diseases and/or coronary heart disease has been assessed, which suggests the validity of this SNP as a putative genetic risk factor for both diseases in the Iraqi population. The Hardy-Weinberg equilibrium of the AIM2 SNP did not deviate and determined the quality of frequencies of genotypes and alleles for each study group. The current study found that periodontal indicators were greater in the AS-P group than in other study groups. The microbiome of plaque secretes a wide range of microbial residues, which in turn accelerates the degradation of periodontium, leading to pocket formation, resorption of bone, and missing teeth by initiating an inflammatory response, which is in agreement with previous studies.16,23,24 Research on the Iraqi population has consistently shown that periodontitis is more common in males than females. This may be attributed to variations in hygienic protocols.25,26 Male individuals exhibit a higher incidence of atherosclerosis than females because of the protective benefits of estrogen hormones in females. Estrogen has numerous influences on fatty acids, oxygen consumption, blood flow, and oxidative characteristics.27,28 Therefore, the males appear to be more susceptible to periodontitis and CHD in the patient groups. The Pearson’s correlation coefficient results of this study indicate that AIM2 genetic variation is meaningfully associated with indicators of periodontal disease severity in periodontal disease patient groups. One possible explanation for this association is the presence of genetic and inflammatory mechanisms that contribute to the progression of periodontal disease and plaque production in atherosclerosis in cardiovascular disease. The present study has several limitations. Popular risk variables for periodontitis and CHD, including cigarette use, high blood pressure, diabetes, and obesity, which might potentially influence or enhance the effects of certain genetic mutations, were deliberately eliminated from the present investigation to prevent bias. Nevertheless, in the presence of various polymorphisms, they may be vulnerable to periodontal and coronary heart diseases. This study is unique in its investigation of the genetic underpinnings of periodontal disease with and without CHD. It had an adequate number of participants, which could be comprehended based on the pilot study to support numerous test adjustments and helped avoid false-positive results commonly found in applicant genes looking at research. Additionally, the results of this case-control study can only be used to establish a link and potential genetic vulnerability in the Iraqi population. To determine whether the AIM2 SNP is applicable to other populations, further experiments are necessary. Considering the correlation between AIM2 genetic substitution and disease severity, our findings not only shed light on disease progression but also suggest a novel approach for treating periodontal disease and coronary heart disease.\n\nAll genomic sequences of AIM2 gene are recorded in National Centre for Biotechnology Information. The access numbers are: LC741258, LC741260, LC741262, LC741264, LC741266, LC741268, LC741270, LC741272, LC741354, LC741355, LC741356, LC741357, LC741358, LC741359, LC741360 and LC741361; the access numbers are available at insert in the gene bank (https://www.ncbi.nlm.nih.gov/genbank/).\n\n\nConclusions\n\nThe presence of the T allele and (GT, TT) genotypes of the AIM2 gene, which reveal a wide range of effects on several traits, corresponds to increased vulnerability to both periodontitis and CHD. This genetic variation plays a role in the development of both the diseases. The presence of the AIM2 genetic variation might potentially disrupt the control of epigenetic processes. This can lead to the production of abnormal AIM2 polypeptides and increase the release of inflammatory mediators in both periodontitis and atheroma formation. The AIM2 SNP was also found to be significantly correlated with indicators of periodontal disease severity. The association between these ailments can be attributed to the presence of shared inflammation and molecular mechanisms in their development. The results of the current study raise issues regarding the validity of this SNP as a putative inherited risk factor for these illnesses in Iraqi males.\n\n\nAuthor contributions\n\nConcept and design: Zina Ali Daily and Batool Hassan Al-Ghurabi data collection, processing, or interpreting: the authors; writing up the manuscript: Zina Ali Daily; revising the work critically for key ideas and analyses of statistics: Zina Ali Daily and Batool Hassan Al-Ghurabi; supervision: Batool Hassan Al-Ghurabi.\n\n\nEthical approval\n\nThe research followed the tenets of the Declaration of Helsinki and its later amendments for human research. The Ethics Committee of college of Dentistry, University of Baghdad approved this study (Ref. 652, 13/09/2022, Project # 652622in 13/09/2022). All participants entered the study after they were received full information about the nature, aims and processes of the study before signing an informed written consent form.\n\n\nPatient consent\n\nAll participant signed an informed written consent form which approved by the Ethics Committee following provision of detailed information about the study and its purpose in a consecutive series manner.", "appendix": "Data availability\n\nFigshare: 1-Data=Zina Ali-Res=F1000 Research.xlsxData for Zina paperAbsent in Melanoma 2 Gene Associated Periodontitis and Coronary Heart Disease, version 2. DOI: https://doi.org/10.6084/m9.figshare.24017892.v2. 29\n\nThis project contains the following underlying data:\n\nThe data contains the recording of periodontal parameters (plaque index (PL), bleeding on probing (BOP), probing pocket depth (ppd) and clinical attachment loss (CAL)) for periodontitis and coronary heart disease patients.\n\nData are available under the terms CC0\n\nFigshare: Data. https://doi.org/10.6084/m9.figshare.24100050.v2. 30\n\nThis project contains the following extended data:\n\nThe data contains the details of age, sex, body mass index (BMI), Aim2 genotypes, clinical parameters for periodontitis and coronary heart disease patients with genetic variation of AIM2 gene.\n\nData are available under the terms CC0.\n\n\nReferences\n\nOrlandi M, Graziani F, D’Aiuto F: Periodontal therapy and cardiovascular risk. Periodontology. 2020; 83(1): 107–124. Publisher Full Text\n\nAbdulkareem AA, Abdulbaq HR, Milward MR: In Vitro Homeostasis of Rat Oral Epithelial Cell Cultures Following Withdrawal of Periodontal Pathogens. Braz. Dent. J. 2020; 31(2): 135–142. PubMed Abstract \| Publisher Full Text\n\nLaine ML, Crielaard W, Loos BG: Genetic susceptibility to periodontitis. J. Periodontol. 2000. 2021; 58: 37–68. Publisher Full Text\n\nHerrera D, Molina A, Buhlin K, et al.: Periodontal diseases and association with atherosclerotic Disease. Periodontology. 2020; 83: 66–89. Publisher Full Text\n\nSaliem SS, Bedea SY, Cooperb PR, et al.: Pathogenesis of periodontitis-A potential role for epithelial mesenchymal transition. Jpn. Dent. Sci. Rev. J. 2022; 58: 268–278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Obaidi MJ, Al-Ghurabi BH: Potential Role of NLRP3 Inflammasome Activation in the Pathogenesis of Periodontitis Patients with Type 2 Diabetes Mellitus. J. Med. Chem. Sci. 2023; 6(3): 522–531. Publisher Full Text\n\nTurer CC, Durmus D, Balli U, et al.: Effect of non-surgical periodontal treatment on gingival crevicular fluid and serum endocan, vascular endothelial growth factor-A, and tumor necrosis factor-alpha levels. J. Periodontol. 2017; 88: 493–501. PubMed Abstract \| Publisher Full Text\n\nAl-Ghurabi BH: The Role of Soluble TLR-2 in the Immunopathogenesis of Gingivitis. Intern. Med. 2021; 28(1): 37–39. Reference Source\n\nWang Y, Liu X, Shi H, et al.: NLRP3 inflammasome, an immune-inflammatory target in pathogenesis and treatment of cardiovascular diseases. Clin. Transl. Med. 2020; 10(1): 91–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchunk SJ, Kleber ME, März W, et al.: Genetically determined NLRP3 inflammasome activation associates with systemic inflammation and cardiovascular mortality. Eur. Heart J. 2021; 42(18): 1742–1756. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Gao J, Huang C, et al.: Roles of AIM2 Gene and AIM2 Inflammasome in the Pathogenesis and Treatment of Psoriasis. J. Front. Genet. 2022; 13: 929162. Publisher Full Text\n\nTonetti MS, Greenwell H, Kornman KS: Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J. Periodontol. 2018; 89(1): S159–s172. PubMed Abstract \| Publisher Full Text\n\nChapple ILC, Mealey BL, Van Dyke TE, et al.: Periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: Consensus report of workgroup 1 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J. Clin. Periodontol. 2018; 45(20): S68–S77. Publisher Full Text\n\nO’Leary TJ, Drake RB, Naylor JE: The plaque control record. J. Periodontol. 1972; 43(1): 38. Publisher Full Text\n\nMühlemann HR, Son S: Gingival sulcus bleeding--a leading symptom in initial gingivitis. Helv. Odontol. Acta. 1971; 15: 107–113. PubMed Abstract\n\nAl-Taweel FBH, Saliem SS, Abd OH, et al.: Assessment of Serum Interleukin-1β and Interleukin-6 Levels in Patients with Chronic Periodontitis and Coronary Heart Disease. Eur. J. Gen. Dent. 2021; 10: 78–83. Publisher Full Text\n\nGita BJ, George AV, Pavithra N, et al.: Dysregulation Of miR-146a by Periodontal Pathogens: a risk for Acute Coronary Syndrome. J. Periodontol. 2019; 90(7): 756–765. PubMed Abstract \| Publisher Full Text\n\nYagnik K, Mahendra J, Kurian VM: The Periodontal-Cardiovascular alliance: Evaluation of miRNA-146a in subgingival plaque samples of chronicperiodontitis patients with and without coronary heart disease. J. Investig. Clin. Dent. 2019; 10: e12442. PubMed Abstract \| Publisher Full Text\n\nTemelli B, Yetkin Z, Savas H, et al.: Circulation levels of acute phase proteins pentraxin 3 and serum amyloid A in atherosclerosis have correlations with periodontal inflamed surface area. J. Appl. Oral Sci. 2018; 26: e20170322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammed HA, Abdulkareem AA, Zardawi FM, et al.: Determination of the accuracy of salivary biomarkers for periodontal diagnosis. Diagnostics. 2022; 12: 2485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarchesan JT, Jiao Y, Moss K, et al.: Common polymorphisms in the IFI16 and AIM2 genes are associated with periodontal disease. J. Periodontol. 2017; 88(7): 663–672. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFigueira MBD, de Lima DS , Boechat AL, et al.: Single-Nucleotide Variants in the AIM2 – Absent in Melanoma 2 Gene (rs1103577) Associated with Protection for Tuberculosis. J. Front. Immunol. 2021; 12: 604975. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaker ZF, Hashem BH: Study the role of proinflammatory and anti-inflammatory cytokines in patients with chronic periodontitis in Iraqi. J. Bagh. College Dentistry. 2012; 24(1): 164–169. Reference Source\n\nAl-Ghurabei BH: Evaluation of serum anticardiolipin antibody, hs-CRP, and IL-6 levels in chronic periodontitis as possible risk factors for cardiovascular diseases. J. Bagh. College Dentistry. 2012; 24(2): 161–165. Reference Source\n\nFadhil R, Al Ghurabi BH, AL-Ghaban NM, et al.: Nuclear factor-kappa B gene Polymorphism and Interleukin-8 in Iraqi population with severe chronic periodontitis. J. Glob. Pharma Technol. 2019; 11(9): 181–186. Reference Source\n\nAbdulkareem AA, Abdulbaqi HR, Nayyef HK, et al.: This retrospective study investigated the consistency between reported chief complaint and periodontal health status of Iraqi patients in relation to age and gender (A retrospective study). J. Bagh. College Dentistry. 2019; 31(2): 65–69. Publisher Full Text\n\nLansky AJ, Ng VG, Maehara A, et al.: Gender and the extent of coronary atherosclerosis, plaque composition, and clinical outcomes in acute coronary syndromes. J. Am. Coll. Cardiol. Img. 2012; 5(3): S62–S72. PubMed Abstract \| Publisher Full Text\n\nDaily ZA, Al-Ghurabei BH, Al-Qarakhli AM, et al.: MicroRNA-155 (miR-155) as an accurate biomarker of periodontal status and coronary heart disease severity: a case–control study. J. BMC Oral Health. 2023; 23: 868. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaily ZA: Data=Zina Ali-Res=F1000 Research, version 2. Figshare. 2023. Publisher Full Text\n\nDaily ZA: zina ali-res (1).xlsx (20.42 kB). Figshare. 2023. Reference Source" }	[ { "id": "304564", "date": "24 Jul 2024", "name": "Athraa Ali Mahmood", "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\nPresent the work clearly and accurately. However, it is better to add a few words about the statistical method within the methods of the abstract and add the value of p in the results of the abstract for further clarification. It is also preferable to add a source to an existing paragraph within the article and explain the reason for that (after assessing five patients with periodontal disease, two calibrating meetings for indicators of periodontal disease severity were obtained within an hour). Sometimes there are many words that have been abbreviated in the research, so it would be preferable to standardize the abbreviation and not mention the whole word again, for example, body mass index (BMI).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "304567", "date": "30 Jul 2024", "name": "Paras Ahmad", "expertise": [ "Reviewer Expertise Periodontal diseases", "periodontitis", "gingivitis", "saliva", "proteins", "genes", "proteomics", "mass spectrometry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 on AIM2 gene polymorphisms and their association with periodontal disease and coronary heart disease (CHD) presents an interesting case-control design.\nHowever, several deficiencies in the methods can be identified:\nIt is not clear how well the control group (healthy participants) is matched with the other groups in terms of age, gender, and other potential confounding factors. Proper matching is crucial to ensure that differences observed are due to the variables of interest (AIM2 gene polymorphisms) rather than other extraneous variables. The clinical assessments of periodontal disease indicators and CHD diagnosis involve subjective components and specialist interpretations. There could be variability in these assessments, and the study does not mention blinding of assessors, which could introduce bias. The study relies on one-way ANOVA and Odds/Ratios without detailed reporting of how these analyses were conducted, including handling potential confounders. Additionally, the use of Pearson correlation coefficients for genetic polymorphism analysis might not be the most appropriate method, given the typically non-linear relationships and categorical nature of genetic data. While the study attempts to control for some confounders by excluding certain participants, it does not appear to account for other potential confounders, such as socioeconomic status, diet, and oral hygiene practices, which could influence both periodontal health and CHD. The focus on a single gene polymorphism (AIM2 G/T rs2793845) might be too narrow, as complex diseases like periodontal disease and CHD are often influenced by multiple genetic and environmental factors. Additionally, the study does not discuss the functional implications of this polymorphism or its interaction with other genetic factors. The study design is cross-sectional, limiting the ability to infer causation or understand the temporal relationship between AIM2 polymorphisms, periodontal disease, and CHD. Longitudinal studies would be more informative in establishing these relationships.\nAddressing these deficiencies would enhance the study’s rigor, reliability, and applicability. Thanks\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/13-810
https://f1000research.com/articles/12-1030/v1	23 Aug 23	{ "type": "Study Protocol", "title": "Uptake of core outcome sets by clinical trialists in China: a protocol", "authors": [ "Ruijin Qiu", "Xiaodan Fan", "Wenhui Wang", "Mike Clarke", "Zhuo Chen", "Shuling Liu", "Paula Williamson", "Hongcai Shang", "Ruijin Qiu", "Xiaodan Fan", "Wenhui Wang", "Mike Clarke", "Zhuo Chen", "Shuling Liu" ], "abstract": "Background: The concept of core outcome sets (COS) has been introduced in China for about 10 years. In recent years, some Chinese researchers also committed to developing COS, though the majority of COS are ongoing. However, there were more than 500 published COS for research in the COMET database by 2020. Whether the disease category of ongoing and completed COS include the burden of disease in China is unclear. In addition, whether the published COS are used by clinical trialists is also unclear. In this research, we would like to investigate if COS are applicable to the burden of disease in China, and to ascertain whether completed COS are used by clinical trialists in China. Methods: The main burden of disease in China will be identified. Then we will search the COMET database to identify if there are ongoing or completed relevant COS research since 2012. Only COS for clinical trials or clinical research will be included. We will extract scopes of published eligible COS, including condition, population, interventions, and core outcomes. Then we will search the Chinese Clinical Trial Registry using disease names for each disease that has a published COS. We will assess the overlap in scope between clinical trials and COS. Then we will conduct an online survey and semi-structured interviews to identify the knowledge and perceptions of COS among primary investigators of included clinical trials. Discussion: This research will fill in gaps between COS and the burden of disease in China. Understanding clinical trialists’ knowledge and perceptions of COS may help dissemination and application of COS in the future. Trial registration: This research is registered in Core Outcome Measures in Effectiveness: https://www.comet-initiative.org/Studies/Details/2563.", "keywords": [ "Core outcome sets", "clinical trialists", "uptake" ], "content": "Background\n\nA core outcome set (COS) is an agreed standardized set of outcomes that should be measured and reported, as a minimum, in all clinical trials in specific areas of health or health care.1 The concept of COS was introduced in China in 2013.2 Chinese researchers have registered more than 80 COS for research in the Core Outcome Measures in Effectiveness Trials (COMET) database by 2022, the majority of them are focused on traditional Chinese medicine (TCM).\n\nA search of the COMET database shows that more than 500 completed COS studies have been published by 2022. The number of COS varies by disease category. The top five disease categories are cancer, rheumatology, neurology, heart and circulation, orthopaedics and trauma.3 However, the number of candidates vary by disease category. In addition, there are more than one COS for some conditions, while no COS exists in some other important conditions. Whether there are gaps between COS research and the burden of disease in China remains unclear.\n\nThere are some benefits to using COS in clinical trials, which include improving the consistency of outcome reporting, increasing the relevance of outcomes measured to decision-makers, and helping to identify potential selective bias. However, previous research has shown that COS uptake is low in most research areas.4,5 Research shows that clinical trialists’ awareness and understanding of COS may facilitate the use of COS.6 However, Chinese clinical trialists’ awareness, understanding and using of COS are unknown. In China, COS research is mainly focused on traditional Chinese medicine. The differences of COS knowledge, awareness, and understanding between TCM trialists and Western medicine trialists are also unclear.\n\nThe aims of this study are to examine: (1) whether the top 25 diseases with the highest burden in China have relevant COS; (2) the use of COS in clinical trials registered in 2021 and 2022 in the Chinese Clinical Trial Registry for these 25 diseases; and (3) views of these clinical trialists on the knowledge, perceptions, and use of COS in relation to choosing outcomes, and whether there are differences between TCM and Western medicine trialists.\n\n\nMethods\n\nThe top 25 causes of disability-adjusted life-years (DALY) in China in 2019 have been identified from the tool of Global Burden of Disease (GBD).7 Two researchers (RQ and XF) will also discuss subtypes of disease according to practice guidelines or textbook of internal medicine if necessary. The initial list of diseases for the study is shown in Table 1.\n\nWe will manually search the COMET database and identify COS for diseases/subtypes in the diseases list. We will identify if there are ongoing or completed relative COS research since 2012, when COS were introduced in China. The study began on April 7th, 2023 and is ongoing.\n\n\n\n(1) The scope appears relevant to a disease in the diseases list;\n\n(2) COS for research that are well developed, meeting existing standards8;\n\n(3) The COS was published after 2012, and the full article can be retrieved.\n\nRQ will identify COS from the COMET database, and XF will check the results. Any discrepancies will be resolved by consensus discussion or by consulting a third reviewer (PW). The COS-STAD recommendation8 will be used to assess the quality of COS by RQ and PW. RQ and PW will discuss all the COS for the 25 causes of DALY and determine which COS are eligible.\n\nWe will search the Chinese Clinical Trial Registry (https://www.chictr.org.cn/historyversionpub.aspx) according to the disease name extracted for the published COS.\n\nInclusion criteria for clinical trials:\n\n(1) Randomised and non-randomised clinical trials;\n\n(2) The objectives of clinical trials are for disease therapy;\n\n(3) The prospective registration time should be in 2021 and 2022.\n\nExclusion criteria for clinical trials: The clinical trials are to assess the mechanisms of effect of interventions.\n\nIf the disease name in the COS is broader, we will further search subtypes of disease. RQ and XF will discuss the list of subtypes of disease, any disagreement will be discussed with a third investigator (HS). XF, WW, ZC, and SL will search for clinical trials from the Chinese Clinical Trial Registry and apply the inclusion and exclusion criteria to identify the eligible clinical trials. RQ will check the results.\n\nInclusion criteria:\n\n(1) An eligible COS exists.\n\n(2) The number of clinical trials with matching scope to an eligible COS is not less than 40.\n\nAfter discussion by MK, PW, HS, and RQ, they believe that a COS developed by Chinese researchers may be significant for Chinese clinical trialists. If there is an eligible COS developed by Chinese researchers, it will be included. If it does not meet the inclusion criteria for comparison of COS with clinical trials, then the prospective registration time for clinical trials will be expanded.\n\nWe will describe the number of ongoing and published COS for the highest burden of disease in China. Full texts will be retrieved for published COS only.\n\nData will be retrieved from previous COMET systematic reviews. Extracted data for COS will include: author, title, publication time, condition, population, intervention, outcomes, and outcome measurement instruments (if applicable).\n\nExtracted data for clinical trials will include: applicant, the applicant’s email and telephone number, the primary investigator’s name, the primary investigator’s email and telephone number, title, applicant’s institutions, study type, disease name, population, intervention, outcomes, and outcome measurement instruments.\n\nFor comparing trial outcomes and COS, only published COS will be used. If there are more than two COS for a specific disease, the differences in scope between these COS will be discussed by two reviewers.\n\nWe will compare the overlap in scope between clinical trials and COS according to the framework used in previous research.9 The framework is shown in Table 2.\n\nWe will calculate the median percentages of outcomes in each clinical trial that were specific matches, general matches, and non-matches with outcomes in each relevant COS. We will analyze the median of the proportion of outcomes that overlap between the COS and clinical trials for each disease. We will also compare the differences between clinical trials of TCM and Western medicine. We will calculate the number of 100% match between clinical trials and COS and look for trends over time.\n\nA survey will be sent to clinical trialists who will conduct a clinical trial that has overlapped scope between the trial and COS. We will identify the primary investigator’s name, email, and telephone number when we extract eligible clinical trials from the Chinese Clinical Trial Registry. We will send a Chinese translation of the COS deemed relevant to the trialists’ trials with questionnaire. When the primary investigators cannot be contacted via emails, the applicants for registration will be approached. We will examine trialists’ knowledge and awareness of COS. An online survey will be sent to clinical trialists, with one of three versions of the survey: 1) clinical trialists who report a full COS in their clinical trials; 2) clinical trialists who report only a few of outcomes in the COS; and 3) clinical trialists who do not report any outcome in the COS.\n\nThe survey will include both closed and open-ended questions. General information, such as gender, age, participants’ role, work experience, geographical area, professional qualification, and educational background will be collected. We will refer the facilitators and barriers identified in previous research, including using of COS and clinical practice guidelines,10,11 to develop open and closed questions to ask all participants. MK, PW, and RQ will discuss the questions in the questionnaires. The survey and other tools will be distributed into Chinese. RQ will translate tools into Chinese and the translation will then be sent to several researchers who have experience in COS research from a Chinese authors' team and we will ask them to give comments. RQ, SL, ZC, and WW will discuss and determine the translation before distribution.\n\nThe structured questions in the survey are shown in extended data.\n\nAt the end of each survey, we will ask the trialists if they would like to attend a semi-structured interview. We will invite at least 30 trialists who agree to be interviewed to attend an in-person or online interview. The COS publication, translated into Chinese, will be sent to them before the interview. The topic guide is as follows:\n\n1. How many years of experience do you have in the design, management, or analysis clinical trials?\n\n2. How many clinical trials did you participate in the design, management, or analysis?\n\n3. When you design a clinical trial, how did you choose outcomes?\n\n4. Do you know what is COS?\n\n5. Are you familiar with COS?\n\n6. Can you define COS?\n\n7. How comfortable you are with English?\n\n8. How did you become familiar with COS?\n\n9. If there is a relevant COS in your research area, do you want to use it? Why?\n\n10. Whether you know the COS that we sent you? Will you use it in your next trial?\n\n11. What kind of facilitators and barriers for you to use a COS in clinical trial?\n\n12. In your opinion, what should be done to improve the use of COS?\n\nData collected from the review of COS and clinical trials, and the survey of trialists will be analysed using descriptive methods. The numbers and percentage of trialists who know the COS and would like to use a COS will be calculated. The trialist’s awareness of, and decisions to search for and use a COS will be analyzed by content analysis.\n\nDissemination\n\nThe finding of this research will be disseminated through conferences, peer-reviewed publication, and social media.\n\n\nDiscussion\n\nCOS have been introduced in China for about 10 years. Chinese trialists’ knowledge and awareness of using COS are important to COS developers in China. This study will provide information on the proportion of trialists in China who registered clinical trials in the Chinese Clinical Trial Registry in the areas with the highest burden of disease, and how the outcomes they chose compare to existing COS.\n\nCOS are very important for reducing heterogeneity of outcome reporting, potential bias, and research waste when they are used in clinical trials. There are hundreds of COS published in the world. Chinese researchers are also developing COS in some health areas, whether there is a gap between COS and the burden of disease in China remains unclear. This research will provide evidence relevant to COS researchers and clinical trialists.\n\n\nEthics and consent\n\nThe project has been approved by the Ethics Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (2023DZMEC-175-01). The ethical approval agreed to not getting written informed consent from the participants, because they are clinical trialists.\n\nTrial registration: This research is registered in Core Outcome Measures in Effectiveness: https://www.comet-initiative.org/Studies/Details/2563.", "appendix": "Data availability statement\n\nNo data are associated with this article.\n\nDANS-EASY; Questionnaire of trialists’ perceptions in COS. https://doi.org/10.17026/dans-ze4-tby3. 12\n\nThis project contains the following extended data:\n\n• Questionnaire of trialists' perceptions in COS(1).pdf. (English version of survey).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe thank the China Scholarship Council for supporting this research. We are grateful to Jamlick Karumbi, from the University of Liverpool for his share on the experience of developing questionnaire.\n\n\nReferences\n\nhttp\n\nZhang L, Zhang J, Chen J, et al.: Clinical research of traditional chinese medicine needs to develop its own system of core outcome sets. Evid. Based Complement. Alternat. Med. 2013; 2013: 202703.\n\nGargon E, Gorst SL, Matvienko-Sikar K, et al.: Choosing important health outcomes for comparative effectiveness research: 6th annual update to a systematic review of core outcome sets for research. PLoS One. 2021; 16(1): e0244878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliamson PR, Barrington H, Blazeby JM, et al.: Review finds core outcome set uptake in new studies and systematic reviews needs improvement. J. Clin. Epidemiol. 2022; 150: 154–164. Publisher Full Text\n\nMatvienko-Sikar K, Avery K, Blazeby JM, et al.: Use of core outcome sets was low in clinical trials published in major medical journals. J. Clin. Epidemiol. 2022; 142: 19–28. PubMed Abstract \| Publisher Full Text\n\nHughes KL, Williamson PR, Young B: In-depth qualitative interviews identified barriers and facilitators that influenced chief investigators' use of core outcome sets in randomised controlled trials. J. Clin. Epidemiol. 2022; 144: 111–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttp\n\nKirkham JJ, Davis K, Altman DG, et al.: Core Outcome Set-STAndards for Development: The COS-STAD recommendations. PLoS Med. 2017; 14(11): e1002447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaldanha IJ, Dodd S, Gorst SL, et al.: More than half of systematic reviews have relevant core outcome sets. J. Clin. Epidemiol. 2021; 136: 168–179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes KL, Williamson PR, Young B: In-depth qualitative interviews identified barriers and facilitators that influenced chief investigators’ use of core outcome sets in randomised controlled trials. J. Clin. Epidemiol. 2022; 144: 111–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCiro Correa V, Helena Lugo-Agudelo L, Camilo Aguirre-Acevedo D, et al.: Individual, health system, and contextual barriers and facilitators for the implementation of clinical practice guidelines: a systematic metareview.\n\nQiu R: Questionnaire of trialists' perceptions in COS. Dataset. DANS. 2023. Publisher Full Text" }	[ { "id": "223685", "date": "30 Nov 2023", "name": "Zehuai Wen", "expertise": [ "Reviewer Expertise Evidence-based medicine research in traditional medicine", "clinical epidemiology", "COS 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\nThis is an exciting study with expected results. As far as I know, this may be the first survey in this field in China. The protocol was clearly reported in terms of the rationale, purpose, and procedure of the study.\n\nIn addition to the literature review, the study also designed a survey for trial investigators, which is a commendable survey to understand further the current situation of knowledge and application of COS among clinical trial investigators in China, which is also conducive to the development and promotion of COS.\n\nFrom the structured questions in the survey, it should be possible to analyze why the investigators used or did not use COS, so it is recommended to consider this analysis in the statistical analysis 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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11295", "date": "12 Apr 2024", "name": "Ruijin Qiu", "role": "Author Response", "response": "Thank you for your comments and useful feedback. From the structured questions in the survey, trialists who searched COS before registration will be asked about the reason why they did not use the COS; their responses will be analyzed by content analysis." } ] }, { "id": "243145", "date": "28 Feb 2024", "name": "Kim Thomas", "expertise": [ "Reviewer Expertise Applied health research", "core outcome sets", "development and validation of outcome instruments" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUptake of core outcome sets by clinical trialists in China: a protocol\n\nThank you for asking for a review of this interesting study protocol. Overall, the protocol is well written.\nThe authors state that the two aims of this protocol are to:\nDetermine whether ongoing and completed COS are relevant to the disease burden in China. Find out whether clinical trialists in China use published COS in their trials.\n\nThey have identified the top 25 disease categories in China, based on the number of disability-adjusted life years (DALYs). They plan to search the COMET database to find ongoing and published Core Outcome Sets (COS) that have been added since 2012, that are relevant to these 25 disease categories. They will also search the Chinese Clinical Trial Registry to identify clinical trials (registered 2021-2022) in the 25 disease categories that list outcomes that match the outcomes in the disease-specific COS. This will give an indication of whether ongoing and completed COS are relevant to the disease burden in China.\nFor further analysis, individual clinical trials will be analysed to see whether an appropriate COS exists for the disease in question. Each trial also has to meet the inclusion criteria of there being at least 40 clinical trials that match to each specific COS. Lead investigators of the selected clinical trials will then be surveyed, and some will be interviewed, on their views of COS.\nThis is an ambitious study that will provide an interesting insight into COS uptake in China. It is actually three studies in one:\n\nMapping of burden of disease in China to published COS. Exploring uptake of COS in trials registered on the Chinese clinical trials registry. Mixed methods study to establish awareness, barriers and facilitators to COS uptake (including both an initial survey and subsequent interview study).\n\nIt might have been helpful to describe these three aspects separately, with specific aims, objectives and methods for each. Not all of the studies aims and objectives are clearly articulated in the existing two aims.\nThe authors suggest that specific COS may be required to evaluate interventions testing Chinese Medicine and that uptake of COS developed in China might be higher than uptake of COS developed internationally. I am a little worried that such a stance goes against the very essence of what COS initiatives are trying to do. Namely to promote a single unified approach to evaluating outcomes in a given condition throughout the world. I am not yet convinced that a COS specific to Chinese Medicine is helpful or required.\nThe approach of focussing on the top 25 diseases by burden of disease in China is interesting and should help to focus the study, although this still represents a lot of work. It is not clear why only COS published since 2012 are included. If a disease category has a gold-standard COS that was published pre-2012 (e.g the work of the OMERACT group), this protocol would count the disease category as having no COS, which seems a shame.\nIt is proposed that the COS identified through a search of the COMET database will be evaluated for quality. Whilst this is a laudable ambition, there was very little detail provided as to exactly how this would be done and what the cut-off for meeting the quality standard might be. Rather than excluding COS based on their quality status, I wonder if it would be more useful to include all identified COS, and to report the quality ratings descriptively. At least this information would then be in the public domain and could avoid wasted effort of people repeating these quality assessments. One of the specified inclusion criteria for study 2 (uptake of COS in clinical trials and studies) required that there be “not less than 40 trials for a given condition”. It might be helpful to add an explanation as to why this was included. If trials are only included in cases where at least 40 of them match a specific COS, the authors are not capturing up to 39 trials per disease category.\nSimilarly, in the section describing data extraction, it might be helpful to signpost to the fact that study 3 will require the contact details of clinical trialist who have been identified in study 2. Otherwise, the reader is left wondering why this information was being extracted from the registry data.\nIn the data extraction section, it states that ‘data will be retrieved from previous COMET systematic reviews’ but it is not clear how this information will be used in the protocol.\nWe have suggested a few minor wording amendments to improve readability:\nBackground\nSuggest amend to: “There are some benefits to using COS in clinical trials, which include improving the consistency of outcome reporting, increasing the relevance of outcomes measured to decision-makers, and helping to identify potential selective outcome reporting bias.”\n\nMethods\nConfusing typo in survey section says ‘A survey will be sent to clinical trialists who will conduct a clinical trial’ – should this be ‘A survey will be sent to clinical trialists who have conducted a clinical trial’?\nIn the survey, there is a question about whether the respondent is a working in Traditional Chinese Medicine, Integrative Medicine or Western Medicine. Integrative Medicine is not mentioned anywhere in the protocol.\nWe wish the authors good luck with conducting this study and look forward to reading the results.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11296", "date": "12 Apr 2024", "name": "Ruijin Qiu", "role": "Author Response", "response": "Reviewer comment 1. This is an ambitious study that will provide an interesting insight into COS uptake in China. It is actually three studies in one: Mapping of burden of disease in China to published COS. Exploring uptake of COS in trials registered on the Chinese clinical trials registry. Mixed methods study to establish awareness, barriers and facilitators to COS uptake (including both an initial survey and subsequent interview study). It might have been helpful to describe these three aspects separately, with specific aims, objectives and methods for each. Not all of the studies aims and objectives are clearly articulated in the existing two aims. Author response 1. The three aims were listed in the final paragraph of the Background section, and the methods for each are described in separate subsections of the Methods section. Reviewer comment 2. The authors suggest that specific COS may be required to evaluate interventions testing Chinese Medicine and that uptake of COS developed in China might be higher than uptake of COS developed internationally. I am a little worried that such a stance goes against the very essence of what COS initiatives are trying to do. Namely to promote a single unified approach to evaluating outcomes in a given condition throughout the world. I am not yet convinced that a COS specific to Chinese Medicine is helpful or required. Author response 2. We did not intend to suggest that, and have included the rationale for including a COS developed by Chinese researchers in the protocol. Reviewer comment 3. The approach of focussing on the top 25 diseases by burden of disease in China is interesting and should help to focus the study, although this still represents a lot of work. It is not clear why only COS published since 2012 are included. If a disease category has a gold-standard COS that was published pre-2012 (e.g the work of the OMERACT group), this protocol would count the disease category as having no COS, which seems a shame. Author response 3. All COS will be analyzed in the mapping of burden of disease in China to COS. A COS published since 2012 would be preferred to one published before 2012 for the analysis of COS uptake if one meets the eligibility criteria. It has been clarified in the methods. Reviewer comment 4. It is proposed that the COS identified through a search of the COMET database will be evaluated for quality. Whilst this is a laudable ambition, there was very little detail provided as to exactly how this would be done and what the cut-off for meeting the quality standard might be. Rather than excluding COS based on their quality status, I wonder if it would be more useful to include all identified COS, and to report the quality ratings descriptively. At least this information would then be in the public domain and could avoid wasted effort of people repeating these quality assessments. Author response 4. We have clarified that we will use patient participation in COS development as a marker of methodological quality, with the rationale provided. Reviewer comment 5. One of the specified inclusion criteria for study 2 (uptake of COS in clinical trials and studies) required that there be “not less than 40 trials for a given condition”. It might be helpful to add an explanation as to why this was included. If trials are only included in cases where at least 40 of them match a specific COS, the authors are not capturing up to 39 trials per disease category. Author response 5. The online survey will be sent to trialists who registered clinical trials which are relevant to a specific COS. We anticipate a 50% non-response rate, thus if the number of trials is too small, it will be difficult to obtain a reasonable spread of perspectives from trialists about the use of the relevant COS. It was felt that responses from 20 trialists would be helpful based on a typical number of participants involved in qualitative research, thus requiring an anticipated 40 trials This rationale has been explained in the methods (2.2.3). Reviewer comment 6. Similarly, in the section describing data extraction, it might be helpful to signpost to the fact that study 3 will require the contact details of clinical trialist who have been identified in study 2. Otherwise, the reader is left wondering why this information was being extracted from the registry data. Author response 6. This has been explained in 2.2.4. Reviewer comment 7. In the data extraction section, it states that ‘data will be retrieved from previous COMET systematic reviews’ but it is not clear how this information will be used in the protocol. Author response 7. Previous COMET systematic reviews, and ongoing inclusion of published COS by COMET, will be used for details of published COS relevant to this work. Data extraction will include the intended use of recommendations, disease category, disease name, population characteristics (age, sex), interventions, geographical locations of COS developers, geographical locations of COS participants will be extracted, and whether patients participated. This has been explained in 2.1.4. Reviewer comment 8. We have suggested a few minor wording amendments to improve readability: Background Suggest amend to: “There are some benefits to using COS in clinical trials, which include improving the consistency of outcome reporting, increasing the relevance of outcomes measured to decision-makers, and helping to identify potential selective outcome reporting bias.” Methods Confusing typo in survey section says ‘A survey will be sent to clinical trialists who will conduct a clinical trial’ – should this be ‘A survey will be sent to clinical trialists who have conducted a clinical trial’? Author response 8. We have made these changes in the manuscript. Reviewer comment 9. In the survey, there is a question about whether the respondent is a working in Traditional Chinese Medicine, Integrative Medicine or Western Medicine. Integrative Medicine is not mentioned anywhere in the protocol. Author response 9. The text has been amended." } ] } ]	1	https://f1000research.com/articles/12-1030
https://f1000research.com/articles/13-808/v1	17 Jul 24	{ "type": "Case Study", "title": "Clean innovation ecosystems: Lifting distressed communities in Appalachia with clean energy", "authors": [ "William Paolillo", "Benjamin Cross", "Charles Zelek", "Donald Wingate", "Adam Berkebile", "Benjamin Cross", "Charles Zelek", "Donald Wingate", "Adam Berkebile" ], "abstract": "The U.S. government has invested in distressed communities in the 21st century but with minimal effect. Regarding income, poverty, joblessness, and vacancy rates, the average distressed zip code in 2018 showed no improvement compared to its standing relative to the average prosperous zip code in 2000. We have discovered the formation of unique business clusters funded by public-private partnerships have the potential to make a difference in lifting distressed communities. Our research of literature and artifacts (photographs, videos, documents, digital media - websites or social media posts) suggests the discovery of a Clean Innovation Ecosystem (CIE). CIE refers to the network of social entrepreneurs, organizations, institutions, and individuals that work together to promote sustainable technologies and practices. As of the 4th quarter of 2023, manufacturing annual run rate construction spending has skyrocketed to over $200 billion. There are another $600 billion of Voltage Valley projects announced that have not yet been built. Over the past two decades, private investment has been between $20 billion and $100 billion annually in U.S. manufacturing infrastructure. Governments are making unprecedented investments in clean energy - which include approximately $400 billion in funding from the Inflation Reduction Act (IRA), $8 billion to establish 6–10 regional Hydrogen Hubs in the U.S., investments in carbon capture, renewable energy technologies, and other investments in clean energy sectors and technologies. All these investments come with the condition that the investment lifts distressed communities. This article explains why investing in Appalachia and geographic regions with similar characteristics will maximize the social benefit of public investment in a Clean Innovation Ecosystem. Our case study covers the Greater Central Appalachian Voltage Valley (GCAVV) – the states of Kentucky, West Virginia, Ohio, Upstate New York, and Michigan, as well as the Central Appalachian region as defined by 56 of the 85 distressed communities of Appalachia.", "keywords": [ "Clean innovation ecosystem", "hydrogen", "social entrepreneurship", "business cluster", "innovation", "renewable energy", "hydrogen hub" ], "content": "Introduction\n\nCarbon dioxide, methane, and nitrous oxide levels in Earth’s atmosphere have skyrocketed to a point unseen in the past 800,000 years. This rapid warming of our planet is triggering a series of catastrophic events, which are severe and often irreversible changes in the Earth’s climate system. These events include rising sea levels, more frequent and intense heat waves, droughts, floods, and other extreme weather events. The world is at a tipping point, and we face urgent challenges requiring bold, innovative solutions. Governments are making unprecedented investments, which include approximately $400 billion in funding from the Inflation Reduction Act (IRA), $8 billion to establish 6–10 regional Hydrogen Hubs in the U.S., and additional investments in carbon capture, utilization, and sequestration, rare earth elements, renewable energy technologies, and a multitude of other investments in clean energy sectors and technologies.1 The Department of Energy defines a Hydrogen Hub as a hydrogen production, storage, and demand cluster.\n\nThe energy sector is a dynamic integrated system, with developments in one sector directly or indirectly impacting numerous others. This is particularly true for the low-carbon energy sector. Many of the technologies targeted have not been deployed at scale, lack the existing infrastructure to support their deployment, and lack established markets for low-carbon products (electricity, low-carbon fuels, etc.) at levels that provide sufficient incentives to justify large-scale investment.2 Clean energy produced by renewable sources and/or employs technology to decarbonize industry and power production - as defined by the Department of Energy for the Hydrogen RFP. An example of decarbonization would be carbon sequestration. Carbon sequestration is a process that captures and stores carbon dioxide in specific geological formations.\n\nThese factors, combined with the historic investments of the IRA, have resulted in forming a unique configuration of stakeholders in which public-private partnerships have formed at a scale never seen. What we are witnessing is, in fact, the creation of a Clean Innovation Ecosystem (CIE). CIE is an extension, an evolution, of the concepts proposed by Michael Porter’s seminal paper “Clusters and the New Economics of Competition,” published in the Harvard Business Review in 1998.3 A thriving CIE will consist of and support a suite of business (industrial) clusters appropriate for a given geographic region, where its natural resources and collaborative efforts can provide a competitive advantage. Therefore, a CIE can be viewed as a collection of sustainable business clusters working together to promote economic development in a specific geographic region and focused on creating a competitive advantage.\n\nPorter defines clusters as not just geographic concentrations of interconnected companies and institutions in a particular field, but as comprehensive ecosystems. These clusters encompass a wide range of linked industries and other entities crucial to competition. They include, for instance, suppliers of specialized inputs such as components, machinery, and services, and providers of specialized infrastructure. Clusters often extend downstream to supply channels and customers, laterally to manufacturers of complementary products, and to companies in industries related to skills, technologies, or standard inputs. Finally, many clusters incorporate governmental and other institutions—such as universities, standards-setting agencies, think tanks, vocational training providers, and trade associations—that offer specialized training, education, information, research, and technical support.\n\nAdditionally, Porter points to the fact that clusters affect competition in three ways:\n\n1. By increasing the efficacy of companies based in the area\n\n2. By driving the direction and pace of innovation, which underpins future productivity growth\n\n3. By stimulating the formation of new businesses, which expands and strengthens the cluster itself\n\nA cluster allows each member to benefit as if it had a larger scale or had joined with others formally-without contractually requiring it to sacrifice its flexibility.3\n\nLis explains that clusters are increasingly recognized as key coordinators within innovation ecosystems, facilitating comprehensive knowledge creation processes.4 The term ecosystem was originally an ecological metaphor used to describe system-level complexities and can be applied in various contexts. An innovation ecosystem is described as a network of interconnected organizations centered around a leading firm or platform, involving participants from both the production and usage sides, and focused on generating new value through innovation. A distinguishing feature of an innovation ecosystem is the significant role that universities or other research institutions play as primary sources of research outputs.4,5\n\nIn the Greater Central Appalachia region, the Voltage Valley business cluster currently being formed provides an excellent example of what can and needs to be done for a business cluster focused on hydrogen, as proposed by the DOE Hydrogen Hub Initiative.6 The region already has other industries that can become business clusters and join the Greater Central Appalachian CIE. Collectively, the business clusters can collaboratively create a thriving CIE that can ensure distressed communities are included. West Virginia, Ohio, Southwestern Pennsylvania, Eastern Kentucky, Western New York, and Michigan (not an Appalachian State but has close economic ties to Central Appalachia) - are experiencing a new industrial revolution centered around integrated circuits (IC), electrification, recycling, and the mining of information with artificial intelligence and machine learning as the region transforms into the GCAVV.6\n\nThese Clean Innovation Ecosystems are leading to the development of Voltage Valleys (Paolillo, 2022), where electric vehicle battery plants, data centers, microchip plants, recycling facilities, and other large power users affiliated with digital transformation or electrification are located. “An example of electrification is powering a car with electricity versus a carbon-based fuel like gasoline”.7 Silicon Valley and electrification are the driving forces behind the construction of these advanced manufacturing facilities and the long-term jobs created by these factories. The Voltage Valley business cluster is different than our last significant business cluster formation.8\n\nFor many years, Silicon Valley on the West Coast of the United States was the epicenter of innovation and wealth creation. Silicon Valley produced groundbreaking solutions and entrepreneurial success stories. Silicon Valley is in the greater San Francisco Bay Area and ignited the growth of venture capital firms – 30% of U.S. venture investment flowed to the greater Silicon Valley in 2023.9 However, a significant part of the Silicon Valley success story involved outsourcing manufacturing to countries like China and Asia, which shifted the U.S. to a service-oriented economy. Unlike the Silicon Valley era, where most manufacturing jobs were outsourced, Voltage Valleys will generate employment opportunities during advanced manufacturing plants’ construction and operation phases. This means jobs for hardworking constructors, makers, and doers who build and create the products that drive the digital age.9\n\nThe Seven Components of the Distressed Community Index\n\nThis is an outline summary of the DOE’s defined key socio-economic indicators used to assess distressed communities.\n\n1. No High School Diploma: This metric represents the percentage of the population aged 25 years and older without a high school diploma or equivalent. It highlights educational attainment levels within the community.\n\n2. Housing Vacancy Rate: This rate measures the percentage of habitable housing that is unoccupied, excluding properties intended for seasonal, recreational, or occasional use. It reflects housing stability and availability in the area.\n\n3. Adults Not Working: This indicator shows the percentage of the prime working-age population (ages 25-54) currently unemployed, highlighting labor market engagement and employment opportunities.\n\n4. Poverty Rate: This metric indicates the percentage of the population living below the poverty line, providing insight into the community’s economic well-being and financial challenges.\n\n5. Median Income Ratio: This ratio compares median household income as a percentage of the metro area’s median household income (or the state median for non-metro areas), offering a comparative view of income levels within the community.\n\n6. Change in Employment: This indicator tracks the percentage change in the number of jobs from 2016 to 2020, reflecting job growth or decline over the specified period.\n\n7. Change in Establishments: This metric measures the percentage change in business establishments from 2016 to 2020, indicating the community’s entrepreneurial activity and business development.\n\nThese indicators, when viewed collectively, provide a powerful and comprehensive overview of the socio-economic conditions in distressed communities. They highlight areas for targeted intervention and support, offering a roadmap for effective community development strategies.\n\nA distressed community faces social, environmental, and economic challenges that do not allow that community to flourish.\n\nThis article explains why investing in Appalachia and geographic regions with similar characteristics will maximize the social benefit of public investment in a Clean Innovation Ecosystem. Our case study encompasses the Greater Central Appalachian Voltage Valley (GCAVV)–the states of Kentucky, West Virginia, Ohio, Upstate New York, and Michigan as well as the Central Appalachian region as defined by 56 of the 85 distressed communities of Appalachia.\n\nSource: https://www.arc.gov/arc-web-and-privacy-policy/ Use or Reproduction of Material from the ARC WebsiteWorks prepared by ARC employees in the scope of their employment are not subject to copyright protection. These works are in the public domain and may be copied and distributed without permission.\n\n\nMethods\n\nWhat factors contribute to lifting distressed communities in a Clean Innovation Ecosystem and have their citizens to flourish?\n\nOur design aims to combine the research methods of literature review and artifacts. The literature review will provide a critical analysis of existing academic literature, while using artifacts will enhance the depth and richness of the data collection process. Artifacts can provide rich, detailed data that may not be available through other data collection methods. Second, artifacts can validate or confirm data collected through other methods, providing a more robust and reliable dataset. Integrating these two research methods, the study aims to explore the research problem from multiple perspectives and validate the findings through triangulation.10\n\nData gathering was done by thoroughly researching relevant literature, including scholarly articles, official reports, industry magazines, and the most recent periodicals. This literature review is useful in discovering the present state of the research subject and getting new ideas for potential solutions. Artifacts can be used as a source of data in three ways: as a means of generating data, as a means of verifying data, or as a means of complementing other data sources.10 Artifacts, whether physical objects like photographs, videos, or documents, or digital media like websites or social media posts, play a pivotal role in our research. They provide crucial contextual information and serve as a strong validation of the findings from other data sources. For instance, photographs and videos offer visual evidence of certain phenomena, while documents and reports provide detailed information and official perspectives. Digital media, such as websites and social media posts, offer real-time data and insights into public opinion and emerging trends.\n\nTo support our research, we have prepared a supplementary section with 17 links to websites and digital articles documenting and detailing the emergence of a Clean Innovation Ecosystem in the Greater Appalachian Voltage Valley. This supplementary section serves as a comprehensive resource, offering a wide range of perspectives and up-to-date information on the development of clean innovation in this region. These resources include government reports, industry analyses, academic studies, and news articles, providing a well-rounded view of the subject matter. We have made a conscious effort to include resources from various stakeholders, ensuring a diverse range of perspectives.11\n\nThe information collected from the literature review and artifacts is studied with content analysis. This method is utilized to locate patterns and subjects in the data, and then use them to form ideas of where government funds should be invested to establish Clean Innovation Ecosystems.12\n\nThe findings of the study encompass a recap of the literature review, artifacts, the results of the content analysis, and the proposals for the maximum and most efficient government investments in forming Clean Innovation Ecosystems.13\n\nThe study’s conclusion includes a review of the research findings. The conclusion will validate the findings through triangulation, comparing the results from the literature review with the insights derived from the artifacts. We will then review the implications for practice and policy, study limitations, and suggestions for potential future research13\n\n\nResults\n\nWhat factors contribute to lifting distressed communities in a Clean Innovation Ecosystem and have their citizens to flourish? See Figure 2 for a graphic representation.\n\nSource: Created by the authors based on the research findings presented in the article, may be used without permission. - article citation of F1000 publication is all that is required for use work or paper.\n\nHypotheses:\n\n1. An established Community Advisory Board (CAB) in the geography funded by a PPP.\n\n2. State and local government agencies work together to leverage federal and private funding.\n\n3. A known and reliable regulatory structure for industrial development\n\n4. Workforce development programs that are broadly supported in geography–to include K -12 STEM programs, community colleges and technical schools, universities, and trade organizations.\n\n5. Clean Innovation Ecosystem consisting of a diverse set of business clusters. For example: the formation of a Regional Voltage Valley Business Cluster growing and characterized by public companies and social entrepreneurship.\n\nOur literature review shows that hypotheses 1–4 have a positive effect on lifting distressed communities. We extend the literature exploring hypothesis 5. The literature and artifacts explain our findings in Hypothesis 5.\n\nThe recent phenomenon of Regional Voltage Valley Business Clusters (RVVBC) will act as a moderator, amplifying the effects of Hypotheses 1-4.\n\nTo lift distressed communities, the following four items are recommended by literature and our research:\n\n1. The establishment of Community Advisory Boards (CAB) within geographic regions funded by a Public-Private Partnership (PPP).14 Communities need to be fully engaged in the entire life cycle of an industrial development project to ensure the safety, security, and economic and environmental impacts are fully understood and the issues are fully addressed. This means the CAB needs to be fully aware of the environmental, social, and governance (ESG) issues related to industrial development in its geographic jurisdiction.14\n\n2. State and local government agencies working together to leverage federal and private funding.15 State and local government agencies must work with economic development organizations (EDOs) in the region to ensure all funding sources are being leveraged to the maximum extent possible. The Minority Business Development Agency (MBDA) is a federal agency that works to promote the growth and competitiveness of minority-owned businesses. By engaging with the MBDA, hydrogen hub developers in Appalachia can access resources and support to help promote diversity and inclusion in their projects, such as by partnering with minority-owned businesses or hiring a diverse workforce.16\n\n3. The Appalachian Regional Commission (ARC) is a federal-state partnership. ARC actively works to promote economic development and enhance the quality of life in the Appalachian region. The ARC offers funding and technical assistance to projects that align with its strategic plan. This plan encompasses supporting social entrepreneurship, building community infrastructure, increasing economic opportunities, and fostering a culture of innovation. By teaming up with the ARC, investors in a hydrogen hub can ensure that their investments are in sync with the needs and priorities of the communities in the region. Moreover, the ARC funds projects prioritizing community benefit initiatives and supporting diversity and minority populations.17\n\n4. A known and reliable regulatory structure for industrial development.18 All legally required regulators must be identified and fully informed about industrial development activities. Agreements will need to be reached as to the roles and responsibilities of each regulator and how any overlap in responsibilities will be addressed. As part of the regulatory structure, each industrial development project must have a single point of contact/entity capable of responding to and addressing regulatory issues.18\n\n5. Workforce development programs that are broadly supported in geography – to include K-12 STEM programs, community colleges and technical schools, universities, and trade organizations.19 For each industrial development project, there needs to be a point of contact/entity to address workforce development issues; the entity should be able to coordinate with existing workforce development programs in the region and provide them a detailed description of their workforce needs, including required training and certification.20 STEM (Science, Technology, Engineering, and Math) education is a critical foundation for workforce development, particularly for high-tech industries such as hydrogen production and fuel cell technology. K-12 STEM programs in Appalachia can provide students with early exposure to STEM fields, cultivate interest and enthusiasm, and help develop a skilled workforce for future high-tech industries.20 Universities in Appalachia can play a vital role in advancing the development and implementation of hydrogen hub technology. By offering specialized education and training programs, universities can help to ensure that the region has a skilled workforce capable of supporting the growth of the hydrogen economy.21 The presence of research institutions like the University of Kentucky’s Center for Applied Energy Research (CAER) underscores the potential for collaboration between academia and industry in the development of hydrogen technology. CAER’s work on hydrogen production and storage is a prime example of the kind of cutting-edge research that universities in the region can conduct to advance the field.21 Moreover, universities can collaborate with industry partners to develop workforce training programs that are tailored to the specific needs of the hydrogen hub industry. By aligning their training programs with the requirements of the industry, universities can help to ensure that the region has a workforce that is well-prepared to meet the demands of the hydrogen economy. This kind of collaboration between industry and academia is key to the successful development and implementation of hydrogen technology.21\n\nWest Virginia University: The university’s National Research Center for Coal and Energy has been actively involved in research related to hydrogen production and utilization. The university also offers a graduate-level certificate program in Alternative Fuels and Vehicle Technologies that covers topics related to hydrogen fuel cell technology.22\n\nVirginia Tech: The university’s Center for Power Electronics Systems has researched fuel cells, including hydrogen fuel cells, as a potential power source for transportation applications. Additionally, the university offers a course on fuel cells as part of its mechanical engineering curriculum.23\n\nOhio University: Russ College of Engineering and Technology research focuses on sustainable energy technologies, including hydrogen fuel cells. The university’s Institute for Sustainable Energy and the Environment has also researched hydrogen production and storage.24 ARC has provided funding to the Voinovich School for Leadership and Public Affairs and to other partners at Ohio University to create two programs to facilitate social entrepreneurism: Social Enterprise Ecosystem (SEE) and one to assist communities with maker spaces and incubators – LIGHTS. The SEE and LIGHTS programs provide no-cost services and access to capital for social entrepreneurs and small businesses in the social sector and early-stage product development.25\n\nMarshall University: The university’s Center for Environmental, Geotechnical and Applied Sciences has researched hydrogen storage and has collaborated with industry partners on the development of hydrogen storage technologies.26\n\nTrade organizations are critical in facilitating communication, collaboration, and workforce development initiatives in the hydrogen hub industry. These organizations have extensive knowledge and experience in the field and can provide a platform for industry professionals to share information and best practices.27\n\nIn addition to promoting collaboration and communication, trade organizations also offer specialized training programs and certifications for professionals in the industry. One such organization is the Appalachian Hydrogen and Carbon Capture Center (AHCCC), based in Morgantown, West Virginia. The AHCCC is a non-profit organization that supports the development of hydrogen and carbon capture technologies in the region. It provides a forum for communication and collaboration between industry stakeholders, research institutions, and government agencies, to advance the development of a hydrogen economy in Appalachia.28\n\nAnother organization that supports hydrogen hubs in the region is the National Energy Technology Laboratory (NETL), which is also based in Morgantown, West Virginia, and has additional locations in Pennsylvania and Oregon. The NETL conducts research and development related to energy technologies, including hydrogen production and storage, and supports the development of a skilled workforce through education and training programs.29\n\nIn addition to these organizations, there are also trade associations at the national level that support the development of hydrogen hubs in Appalachia. The National Hydrogen Association (NHA) and the Fuel Cell and Hydrogen Energy Association (FCHEA) are two such organizations. These associations provide a platform for industry stakeholders to communicate and collaborate on workforce development initiatives, offer specialized training programs and certifications for professionals in the industry, and advocate for policies that support the growth of the hydrogen economy.30,31\n\nRicket et al.32 posit the emergence of an ecosystem that is a convergence of the Social Entrepreneurial Ecosystem (SEE) and Community Sustainability. In this model, social entrepreneurs use their business for good, not just to make a profit but to positively impact the triple bottom line profit—people, planet, and profit. This is a departure from the “urban-centric economic policies [that] have historically used rural areas as “sacrifice zones”: areas environmentally degraded to produce goods (and human capital) necessary to feed, literally and figuratively, urban centers.33–36 A driving force for this change is investment by government agencies that have set a criterion for the award – there must be a positive socioeconomic effect on distressed communities. Public sector investment rules, combined with a new accountability of the economic development in the private sectors, are pushing the normative business process towards social entrepreneurism.37\n\nA way we have seen social entrepreneurship take hold in other rural areas is the Mondragon Cooperative.38 The Mondragon Cooperative Model39 has over 80,000 people with sales in more than 150 countries. It is a worker-owned cooperative, meaning the employees collectively own and manage the business. The model is named after the town in the Basque country of Spain, where the first cooperative was established. In this model, the employees are the owners and decision-makers of the business. They elect a board of directors, who are responsible for managing the day-to-day operations of the business. The board of directors is accountable to the general assembly, which comprises all the worker-owners.39\n\nThe Mondragon Cooperative Model has several distinctive features. One of these is its commitment to social responsibility. The model emphasizes the importance of creating sustainable jobs, promoting social justice, and contributing to the local community.39 Mondragon believes that profitability allows enterprises and ecosystems to afford their chosen values. Mondragon solidarity brings “ordinary people together achieving extraordinary things”.39 Another key feature of the model is its emphasis on education and training. The Mondragon cooperatives have established a network of education and training centers, which provide workers with the skills and knowledge they need to succeed in their jobs and advance within the cooperative.39\n\nJohn Elkington is a sustainability thought leader who introduced the concept of the “triple bottom line,” which considers not just financial profit but also social and environmental impact.40 A Clean Innovation Ecosystem is an interconnected network of organizations, individuals, and resources that work together to develop and deploy sustainable solutions to environmental challenges. The triple bottom line profit advises the Clean Innovation Ecosystem and is specific in its approach to solving the existential threat of climate change – deploying sustainable solutions to environmental challenges.40 The literature guides many models for successful innovation.41\n\nTo maximize social benefit for federal, state, and local agencies in a Clean Innovation Ecosystem, the success of the Clean Energy Ecosystem is not in a model, but in the execution of the relationships. It is essential to foster collaboration, innovation, and equity while setting clear goals and standards and regularly monitoring progress and adapting strategies as necessary.42\n\nThe National Science Foundation suggests that the ’Valley of Death’ for Innovation Hubs is not just a phase, but a critical one. It’s a phase where research funding and public investments cease before commercial success has been established to ensure a sustainable enterprise. The literature suggests that cultivating successful relationships must be intentional and practiced before the Regional CIE (Centers for Innovation Excellence) location receives research funds or resources.43\n\nThe innovation ecosystem comprises various actors, entities, and intangible elements. These intangible elements include the complex relationships and interactions that gradually transform the steep, formidable Valley of Death into a more navigable and supportive Challenge Basin, facilitating smoother progress and greater collaboration within the ecosystem.\n\nInvestors use environmental, social, and governance criteria to evaluate the societal impact and sustainability of companies. ESG criteria include factors like a company’s carbon footprint, diversity and inclusion policies, employee relations, and ethical business practices. ESG is used to evaluate a company’s short- and long-term risks, which directly affect the company’s stock price and return on shareholder equity.44 Net Zero, Fortune 500, and ESG are all related to the growing global concern about sustainability and responsible corporate behavior. The European Union, United Kingdom, and United States are all in the process of developing the mandatory reporting requirements for ESG and expect to have them in place by 2024.44\n\n’Net zero’ is a term used to describe a state where the amount of greenhouse gas emissions released into the atmosphere is balanced by the amount removed from the atmosphere. The goal is to achieve this balance by reducing emissions as much as possible and compensating for any remaining emissions through methods like carbon capture and storage or reforestation. In simpler terms, it is like a scale where the weight of emissions on one side equals the weight of emissions removed on the other, resulting in a balance.44\n\nScope 1, 2, and 3, as defined by the World Economic Forum 2022, are terms used to categorize the different sources of greenhouse gas (GHG) emissions from a company or organization. These scopes are defined by the Greenhouse Gas Protocol, which is a widely used international standard for GHG accounting.44\n\n• Scope 1 emissions: These are direct emissions from sources that are owned or controlled by the company, such as emissions from the combustion of fuels in company-owned vehicles or emissions from company-owned facilities.44\n\n• Scope 2 emissions: These are indirect emissions that result from the generation of electricity, heating, or cooling that the company consumes. These emissions are associated with generating electricity purchased by the company from utilities.44\n\n• Scope 3 emissions: These are indirect emissions that result from activities outside of the company’s operations, such as emissions from the production of raw materials, transportation of products, and the use and disposal of products and services that the company provides.44\n\nTo achieve net-zero emissions, companies must first measure and understand their emissions in all three scopes. Then, they must set targets to reduce emissions and develop a plan to achieve them. The plan should include strategies to reduce energy consumption, switch to renewable energy sources, and develop more sustainable supply chains. Companies must also engage with suppliers, customers, and other stakeholders to encourage them to adopt sustainable practices.\n\nIn summary, understanding and addressing emissions across all three scopes is essential for companies to achieve net-zero emissions and contribute to global sustainability efforts. The relationship between these concepts is that achieving net-zero emissions requires significant changes in how companies operate. The Fortune 500 companies, as major players in the global economy, have a crucial role in achieving net-zero emissions and sustainable development. Investors are also increasingly interested in companies that prioritize ESG considerations, and those that do are likely to be more successful.\n\nThe Intergovernmental Panel on Climate Change (IPCC) has identified 2050 as the deadline for achieving net-zero emissions. To limit global warming to 1.5°C above pre-industrial levels, many countries and companies have set ambitious intermediate targets for 2030 and 2040 to achieve net-zero emissions by 2050. Intel has pledged to achieve net zero by 2040. Companies must lay out their plans and report on their progress to targets.44\n\n\nCase study and discussion\n\nIn an era where the world is facing pressing challenges related to climate change and economic transformation, a shining light is emerging in the heart of America - the Voltage Valley. This region, Greater Central Appalachia encompassing states such as Kentucky, West Virginia, Ohio, Upstate New York, Tennessee, Michigan, and the Central Appalachian communities, is poised to become a driving force behind saving the planet and reinvigorating American manufacturing. Voltage Valley represents a new type of business cluster, one that supports electric vehicle battery plants, data centers driven by artificial intelligence and machine learning, integrated circuit chip manufacturing, recycling facilities, and other large-scale endeavors related to digital transformation and electrification. The Voltage Valley created in the CIE helps pave the way for a more sustainable future.45\n\nSource: Ohio University PORTSfuture Program, funded by a grant from the US Department of Energy Office of Environmental Management Portsmouth/Paducah Project Office. This has been reproduced with permission of Ohio University.\n\nThe leading indicator of the Voltage Valley revolution is the surge in manufacturing construction spending. Over the past two decades, the United States has invested between $20 billion to $100 billion annually in manufacturing infrastructure – a yearly average spending of approximately $60 billion annually. As of July 2023, manufacturing annual run rate construction spending has skyrocketed to over $200 billion. Another $600 billion of Voltage Valley projects are in the U.S. as of the third quarter of 2023.45\n\nFor many years, Silicon Valley on the West Coast of the United States was the epicenter of innovation and wealth creation. It produced groundbreaking solutions and entrepreneurial success stories and ignited the growth of venture capital firms primarily concentrated in the San Francisco Bay Area. However, a significant part of the Silicon Valley success story involved outsourcing manufacturing to countries like China and Asia, which shifted the U.S. economy towards a service-oriented one. Unlike the Silicon Valley era, where most manufacturing jobs were outsourced, Voltage Valleys generated employment opportunities during advanced manufacturing plants’ construction and operation phases. This means jobs for hardworking makers and doers, individuals who build and create the products that drive the digital age.45\n\nThe story of Voltage Valley is not confined to a specific city; it is a phenomenon occurring in regions that were once at the forefront of the Industrial Revolution, such as Greater Appalachia. As the 21st century unfolds, we are witnessing the birth of a new industrial revolution – GCAVV the Voltage Valley Revolution.45\n\nOur case study, as documented by the artifacts, focuses on the Greater Central Appalachian Voltage Valley (GCAVV) forming in Ohio, West Virginia, Kentucky, western Pennsylvania, upstate New York, and parts of Michigan (Figure 3). We have named this region the Greater Central Appalachia Triangle as it spans from Central Appalachia to the Great Lakes of New York, Ohio, and Michigan. The combined GDP of this geography across these six states is approximately $2 trillion, the equivalent of a top 8 country in the world, between Italy (8) and France (7).46 Once we have set the economic conditions of the Greater Central Appalachian Voltage Valley, we will discuss the 56 distressed counties in Central Appalachia, situated within the tri-state region of Ohio, West Virginia, and Kentucky. The 13-state region of Appalachia is among the most economically distressed in the country, including 82 distressed counties. As shown in Figure 1, the Appalachian Regional Commission has assigned the “distressed area” designation to many counties; 56 of the 82 counties were identified as distressed areas.47\n\nThe GCAVV region is experiencing a new industrial revolution centered around integrated circuit chips, electrification, and information mining with artificial intelligence and machine learning as the region transforms into the Greater Appalachian Voltage Valley. The construction of these advanced manufacturing facilities and the long-term jobs created by these factories can potentially grow the defined region’s economy. The actual factory locations are in or around these distressed counties. The government investment criteria to impact distressed communities and this economic surge provides a generational opportunity to lift the people in these geographies. Maximize the social impact of government investment. This is a review of the artifacts we found in our study.48\n\nThe construction value of the defined region’s new unbuilt Greater Central Appalachian Voltage Valley is more than $50 billion over the next five years for this region. According to the Associated Builders and Contractors, every additional billion dollars spent in a region contributes 6,300 additional construction jobs. Assuming an incremental construction build of $10 billion over the next five years, that is an incremental 63,000 construction workers in the Greater Appalachian Voltage Valley projects.49\n\nThe General Motors 2.8 million square feet gigacity Ultium battery plant Gigafactory started operation in Lordstown, Ohio, in August 2022. Construction of the GM Gigafactory started in 2020, providing 5,000 construction jobs and over 2,000 operation jobs at the factory. The cost was over $2 billion. Since 2020, in the Greater Appalachian Voltage Valley, there have been many project announcements of similar scope and community benefit—a Ford electric vehicle plant in Avon Lake, Ohio, two Ford EV battery plants in Glendale, Kentucky, Cleveland-Cliffs Steel investments in Toledo, Nucor Steel in Mason County (classified at risk), West Virginia, Honda EV Battery Plant and Innovation Center in Marysville, Ohio, Cirba Solutions lithium battery recycling in Lancaster, Ohio, and Li-Cycle in Rochester, New York. When fully operational, these two plants will recycle enough lithium ion to make over 250,000 EV car batteries. Another example is Hemlock Semiconductor’s silicon refinery in Lansing, Michigan, which will create 170 permanent jobs and supply polysilicon to the semiconductor and solar industries. Integrated chip manufacturers are opening Intel facilities outside of Columbus, Ohio, and Micron facilities located in the greater Syracuse, New York area. The cost of these facilities is $20 billion and $50 billion, respectively.50\n\nAll the companies establishing their operations in this region are doing so because of access to reliable power and transmission lines. Voltage Valleys need reliable, resilient, and sustainable power to drive advancements in artificial intelligence, blockchain, machine learning, faster IC chips, and robots used in manufacturing, recycling, and product manufacturing that support electrification.51\n\nMoreover, each of these companies has net-zero carbon goals for 2050 based upon ESG plans. The Greater Central Appalachian Voltage Valley ecosystem presents a unique opportunity for the reshoring of advanced industries. The clustering of advanced manufacturing in this region would allow companies access to sustainable power and clean industrial inputs.52\n\nOne of the most vital assets of Central Appalachia is its people; they are “makers” and possess the know-how and the “can-do” spirit needed to manufacture products at scale.53 The makers and the region’s already strong manufacturing base and growing Voltage Valley business cluster provide an excellent foundation upon which to make American manufacturing great again. Advanced manufacturing also requires access to trains, ports, feedstocks, and water. These needs are precisely why the Ohio River Valley has historically been a manufacturing powerhouse.54\n\nWe anticipate high-value, energy-intensive manufacturers making sizeable investments in the Greater Appalachian Voltage Valley to keep up with market demands. The development of these new facilities will require the production of silicon, polymer, and glass inputs essential for the booming electric vehicle, semiconductor, and photovoltaic industries in the area. Fortunately, the Ohio River Valley has the proven ability to take on large energy-intensive projects, and it possesses many assets that can be utilized. In solar manufacturing, Illuminate USA announced a $600 million project to establish a solar panel manufacturing factory in Pataskala, Ohio. The development is expected to create 850 jobs in March of 2023.55\n\nWhen the Piketon Gaseous Diffusion plant opened in 1952, it consumed 5% of all electricity consumption in the United States. At its peak, it used up to 40 million gallons of water each day for cooling. After a decade of inactivity, the Department of Energy decommissioned the facility in 2011. For the first stage of recommissioning, 248 acres of the site will be revitalized by Trillium H2 Power.56\n\nThis $1.5 billion endeavor includes 250 megawatts of power generation capacity and carbon sequestration. The power plant provides 500 metric tons of clean hydrogen daily, which will be utilized for the primary production of silicon and green ammonia, as well as powering a 65-acre greenhouse farm with the waste heat from the power plant.56\n\nThe building and operation of these facilities will require the use of a digital twin as well as project teams that use human-machine collaboration. The Trillium integrated manufacturing facility in Piketon, Ohio, intends to use a digital twin to design, build, and operate the facility. The way it will work is that the architects and designers create a 3D model of the Piketon project – the digital twin. This requires the participants in the project to collaborate and the digital model - Human-machine collaboration. As the participants go through the process of human-machine collaboration, the region’s digital capability grows.56\n\nIn our interviews with Trillium H2 Power officials, they are looking at the Mondragon system of operation and a citizen share model. The farm’s production will be owned by the individuals who work at the co-op. The coop will target the sale of the garden’s fresh vegetables in the local community. The coop sells the surplus in the surrounding cities if there is an incremental product. In the Mondragon model, profit is good; this cooperative wants to take it a step further, creating a citizen share. A citizen share would be 15% of the profits distributed to the employee’s non-profit of choice.56\n\nIn the hills of Kentucky, the world’s most invaluable fossil fuel deposits are not too far away. Kentucky’s Blue Gem Coal seam contains ultra-low sulfur coking coal, which is highly sought after by semiconductor manufacturers. This feedstock is mixed with raw silica and put into arc furnaces to make 99.9% pure silicon for semiconductor production. Ferro’s global arrangement to supply silicon from its Beverly, Ohio, foundry to the Renewable Energy Corporation (REC) plant in Montana speaks to the length’s companies are willing to go to get premium feedstocks.57\n\nHemlock Semiconductor is investing $375 million in a silicon refinery in Lansing, Michigan. Currently, the United States produces 310,000 metric tons of metallurgical grade silicon each year, split between REC and Hemlock. Owing to the surging number of onshoring projects for semiconductor and solar manufacturing, U.S. silicon demand is predicted to double in the coming years. In this case, the Appalachian Triangle and Pennsylvania offer an optimal clean manufacturing ecosystem to produce additional metric tons of metallurgical-grade silicon. Nucor Steel announced a 3 million ton of steel facility in February 2023.58\n\nOn the shores of Lake Erie, Cleveland-Cliffs, the most extensive producer of flat stock steel and iron ore in North America, has built a steel manufacturing plant in Toledo, responsible for 25% fewer carbon dioxide emissions per ton of steel. This facility uses clean hydrogen to reduce iron oxide instead of coal. In Ohio alone, nearly 30,000 acres of solar power plants are either being developed or assessed. This could bring about comprehensive decarbonization throughout the steel manufacturing supply chain of the Greater Central Appalachian Voltage Valley.59\n\n\nConclusions\n\nHydrogen Hubs, carbon capture utilization and sequestration, rare earth elements, renewable energy technologies, and many other investments in the clean energy sectors and technologies can power Clean Innovation Ecosystems. The level of private investment in the GCAVV is unprecedented, as analyzed by the artifacts. Our research points to the positive nature of how a forming RVVBC will moderate or amplify the benefits of federal government investment - uplift distressed communities. The artifacts also show that RVVBC are characterized by social entrepreneurs and major corporations committed to triple bottom line profit. The values of the social entrepreneurs and major corporations with stated ESG goals, tax, and government-guaranteed loan programs compel companies to develop ways to uplift distressed communities. The GCAVV has the opportunity to serve as a case study of what will work and what will not work in a CIE powered by hydrogen and other clean energy technologies. Monitoring the environmental and social landscape, as well as measuring the financial results of those participating in the GCAVV, will value insights on how to lift distressed communities with investments in clean energy.60\n\n\nFuture research\n\n\n\n1. A quantitative multivariant equation model to test the validity of all antecedents.\n\n2. Quantitative study at 5-year intervals to determine best practices and evaluate performance.\n\n3. Compare the European Union’s vs the US’s approach to creating a clean energy-powered CIE.\n\n\n\n1. The ability to obtain data for the complete model of what lifts a distressed community.\n\n2. The ability to obtain valid data on the business clusters that make up the CIE and quantitively verify their individual and collective impact.\n\nIn addition to future research opportunities, there are other important considerations for the development and operation of Clean Innovation Ecosystems in Greater Central Appalachia. One consideration is the identifying and inventorying of assets available in the region, particularly of former fossil fuel sites and their infrastructure that can be repurposed and built back better as part of a CIE. Another key area for research is identifying appropriate business clusters for CIE and how to establish these clusters in the region. It is also important to identify the obstacles and their mitigation options for developing a conducive regional business environment in which a CIE can be established and flourish. Additional Research should explore how to create Community Advisory Boards (CABs) across governmental boundaries and whether multiple CABs can exist across the CIE region, and if so, how can they be coordinated. Finally, to promote public buy-in and support, it is essential to develop effective ways to educate and inform the public of a CIE’s development and its impact on the community, especially in the distressed communities.61\n\n\nAuthor contributions\n\nConceptualization, B.P., B.C., D.W., A.B., and C.Z.; Definitions of Voltage Valley and Clean Innovation Ecosystem, B.P.; Formal analysis, W.P., B.C., and C.Z.; Methodology, B.P.; Original draft preparation, B.P., B.C., and C.Z.; Refinement of regional business cluster concept, B.C.; Resources, B.P.; Validation, B.P., B.C., D.W., and C.Z.; Writing of report, W.P.; Writing of introduction and of literature review for hypotheses 1–4, B.C., A.B., and C.Z.; Writing discussion B.P. & D.W.; Writing about future research, B.C. All authors have read and agreed to the published version of the manuscript.\n\n\nEthics and consent\n\nEthical approval and consent were not required.", "appendix": "Data availability statement\n\nThis study is based on secondary sources. All data supporting the findings are included in the manuscript and its supplementary materials. No underlying data are associated with this article.\n\nThe supplementary materials provide comprehensive documentation of the sources, including hyperlinks to government reports, industry analyses, academic studies, and news articles relevant to the Clean Innovation Ecosystem in the Greater Appalachian Voltage Valley. These resources offer a diverse range of perspectives and up-to-date information on the subject matter. For additional details or specific data inquiries, readers are encouraged to consult the supplementary materials provided.\n\nThe artifacts used in the research can be found on the websites mentioned below:\n\n1. Brookings Institution Report on Distressed Communities\n\n2. Community Voice and Power Sharing Guidebook\n\n3. Community Advisory Board Toolkit\n\n4. Business Strategy and the Environment\n\n5. Why State and Local Relationships Matter\n\n6. Economic Policy Institute Report\n\n7. Personal View: Ohio is Winner in Race to Create Voltage Valleys\n\n8. A Typology of Social Entrepreneurs\n\n9. Economic Impacts of Coal-Fired Power Plants\n\n10. Houston Region Poised to Become Global Clean Hydrogen Hub\n\n11. Innovation Ecosystems: A Conceptual Review\n\n12. Flourishing: The New Spirit of Business Enterprise\n\n13. Doing Interpretive Research\n\n14. The Importance of Workforce Development\n\n15. Working with Value: Industry-specific Approaches to Workforce Development\n\n16. Center for Applied Energy Research (CAER)\n\n17. National Research Center for Coal and Energy\n\n\nAcknowledgments\n\nThis article is informed by the past 13 years of ongoing work carried out by Ohio University’s Voinovich School of Leadership and Public Service PORTSfuture Program. The program is planning for, developing, and pursuing the redevelopment of a federal facility in southern Ohio into a decarbonized energy production and sustainable manufacturing complex. This effort will bring economic stability and prosperity back to the communities in the region while also addressing many national priorities related to energy independence, reshoring of vital supply chains, advancing the clean energy economy, and combatting the climate crisis. PORTSfuture is carried out in partnership with local, regional, national, and federal entities, organized labor, private industry, and many other site stakeholders. Program activities include creating public-private partnerships, community outreach and engagement, data analyses and geographic information systems, economic impact analysis, workforce analysis, K-12 STEM education, and conducting applied environmental work, among other items. The PORTSfuture Program is funded by a grant from the US Department of Energy Office of Environmental Management Portsmouth/Paducah Project Office.\n\n\nReferences\n\nInflation Reduction Act: U.S. Department of Energy.Reference Source\n\nU.S. Department of Energy: Hydrogen Hubs.Reference Source\n\nPorter ME: Clusters and the new economics of competition. Harv. Bus. Rev. November–December 1998; 76(6): 77–90. PubMed Abstract\n\nLis AM, McPhillips M, Lis A: Sustainability of Cluster Organizations as Open Innovation Intermediaries. Sustainability. 2020; 12(10520). Publisher Full Text\n\nAsplund F, Björk J, Magnusson M, et al.: The genesis of public-private innovation ecosystems: Bias and challenges. Technol. Forecast. Soc. Chang. 2021; 162: 120378. Publisher Full Text\n\nDOE Hydrogen Hub Initiative: U.S. Department of Energy.Reference Source\n\nPaolillo B: Personal view: Ohio is winner in race to create voltage valleys. Crain’s Business Cleveland; April 16, 2022. Reference Source\n\nLis AM, McPhillips M, Lis A: Sustainability of Cluster Organizations as Open Innovation Intermediaries. Sustainability. 2020; 12(10520). Publisher Full Text\n\nSilicon Valley venture investment statistics. http\n\nWalsham G: Doing interpretive research. Eur. J. Inf. Syst. 2006; 15(3): 320–330. Publisher Full Text\n\nSupplementary materials documenting Clean Innovation Ecosystem. http\n\nContent analysis methodology. http\n\nProposals for government investments in Clean Innovation Ecosystems. http\n\nArnos D, Kroll E, Jaromin E, et al.: Tools and resources for project-based community advisory boards. Community Voice and Power Sharing Guidebook. October 2021. Reference Source\n\nLiu A, Rezk P: Why state and local relationships matter to national prosperity: A case for economic collaboration. Brookings Institute; January 19, 2023. Reference Source\n\nU.S. Department of Commerce: Minority Business Development Agency (MBDA).2021. Reference Source\n\nAppalachian Regional Commission: About ARC.2021. Reference Source\n\nOrganisation for Economic Co-operation and Development (OECD): Regulatory Policy and the Road to Sustainable Growth.2010. Reference Source\n\nPerez-Johnson I, Holzer H: The Importance of Workforce Development for a Future-Ready, Resilient, and Equitable American Economy. American Institute for Research, Workforce Development and Economic Mobility/Prosperity Team; April 2021. Reference Source\n\nBackes B, Holzer HJ, Velez EE: Is it worth it? Postsecondary education and the labor market outcomes for the disadvantaged. IZA J. Labor Policy. 2015; 4(1): 1–29. Publisher Full Text\n\nUniversity of Kentucky: Center for Applied Energy Research (CAER).2022. Reference Source\n\nWest Virginia University: National Research Center for Coal and Energy.2022. Reference Source\n\nVirginia Tech: Center for Power Electronics Systems (CPESC).2022. Reference Source\n\nOhio University: Institute for Sustainable Energy and the Environment. Russ College of Engineering and Technology; 2022. Reference Source\n\nJolley GJ, Khalaf C, Michaud G, et al.: The economic, fiscal, and workforce impacts of coal-fired power plants in Appalachian Ohio. Reg. Sci. Policy Pract. 2019; 11: 403–423. Publisher Full Text\n\nMarshall University: Center for Environmental, Geotechnical and Applied Sciences (CEGAS).2022. Reference Source\n\nAppalachian Hydrogen and Carbon Capture Center: About the AHCCC.2022. Reference Source\n\nNational Energy Technology Laboratory (NETL): NETL locations: Morgantown, West Virginia. U.S. Department of Energy; 2021. Reference Source\n\nNational Hydrogen Association (NHA): About the National Hydrogen Association.2022. Reference Source\n\nFuel Cell and Hydrogen Energy Association: Certified fuel cell technician program.2022. Reference Source\n\nRicket AL, Knutsen FB, Jolley GJ, et al.: Appalachian social entrepreneurship ecosystem: A framework for rural development. Community Dev. 2022; 54: 315–336. Publisher Full Text\n\nCatte E: What You Are Getting Wrong about Appalachia. Belt Publishing; 2018.\n\nLerner S: Sacrifice Zones: The Front Lines of Toxic Chemical Exposure in the United States. MIT Press; 2010.\n\nOhio River Valley Institute: Appalachia’s natural gas counties: Contributing more to the U.S. economy and getting less in return. Ohio River Institute; 2021. Reference Source\n\nTheobald P: Education Now: How Rethinking America’s Past Can Change Its Future. Routledge; 2009.\n\nClamp C, Peck M: Humanity at Work & Life: Global Diffusion of the Mondragon Cooperative Ecosystem Experience. Oak Tree Press; 2023.\n\nMondragon Corporation: Mondragon.2022. Reference Source\n\nElkington J: Cannibals with Forks: The Triple Bottom Line of 21st Century Business. New Society Publishers; 1998.\n\nGrandstrand O, Holgersson M: Innovation ecosystems: A conceptual review and a new definition. Technovation. February–March 2020; 90-91: 102091–102098. Publisher Full Text\n\nJackson DJ: What is an innovation ecosystem? National Science Foundation; 2011. Reference Source\n\nFink L: The power of capitalism. Blackrock; 2022. Reference Source\n\nAppalachian Regional Commission: Classifying economic distress in Appalachian counties. ARC.gov; 2022. Reference Source\n\nKnowles AK: The making of ethnic capitalists: Welsh iron-makers in southern Ohio. University of Wisconsin–Madison; 1993\n\nPaolillo B, Murray M: Personal view: Here’s why Ohio is poised for decades of job growth. Crain’s Business Cleveland. July 17, 2022. Reference Source\n\nGrandstrand O, Holgersson M: Innovation ecosystems: A conceptual review and a new definition. Technovation. February–March 2020; 90-91: 102091–102098. Publisher Full Text\n\nVelt H, Torkkeli L, Laine I: Entrepreneurial Ecosystem Research: Bibliometric Mapping of the Domain. Journal of Business Ecosystems. 2020; 1: 43–83. Publisher Full Text\n\nAutio E, Thomas L: Innovation ecosystems. The Oxford Handbook of Innovation Management. Oxford, UK: Oxford University Press; 2014; 204–288.\n\nXu Z, Maas G: Innovation and Entrepreneurial Ecosystems as Important Building Blocks. Transformational Entrepreneurship Practices. Springer International Publishing; 2019; pp. 15–32.\n\nVelt H, Torkkeli L, Laine I: Entrepreneurial Ecosystem Research: Bibliometric Mapping of the Domain. Journal of Business Ecosystems. 2020; 1: 43–83. Publisher Full Text\n\nAutio E, Thomas L: Innovation ecosystems. The Oxford Handbook of Innovation Management. Oxford, UK: Oxford University Press; 2014; pp. 204–288.\n\nXu Z, Maas G: Innovation and Entrepreneurial Ecosystems as Important Building Blocks. Transformational Entrepreneurship Practices. Springer International Publishing; 2019; pp. 15–32.\n\nWalsham G: Doing interpretive research. Eur. J. Inf. Syst. 2006; 15(3): 320–330. Publisher Full Text\n\nRicket AL, Knutsen FB, Jolley GJ, et al.: Appalachian social entrepreneurship ecosystem: A framework for rural development. Community Dev. 2022; 54: 315–336. Publisher Full Text\n\nCatte E: What You Are Getting Wrong about Appalachia. Belt Publishing; 2018.\n\nLerner S: Sacrifice Zones: The Front Lines of Toxic Chemical Exposure in the United States. MIT Press; 2010.\n\nOhio River Valley Institute: Appalachia’s natural gas counties: Contributing more to the U.S. economy and getting less in return. Ohio River Institute; 2021. Reference Source\n\nTheobald P: Education Now: How Rethinking America’s Past Can Change Its Future. Routledge; 2009.\n\nClamp C, Peck M: Humanity at Work & Life: Global Diffusion of the Mondragon Cooperative Ecosystem Experience. Oak Tree Press; 2023.\n\nMondragon Corporation: Mondragon.2022. Reference Source\n\nElkington J: Cannibals with Forks: The Triple Bottom Line of 21st Century Business. New Society Publishers; 1998.\n\nGrandstrand O, Holgersson M: Innovation ecosystems: A conceptual review and a new definition. Technovation. February–March 2020; 90-91: 102091–102098. Publisher Full Text" }	[ { "id": "310040", "date": "21 Aug 2024", "name": "Tyler Norris", "expertise": [ "Reviewer Expertise Energy system modeling", "Techno-economic and market analysis", "Statistical analysis", "Machine learning" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents a case study on how public-private partnerships (PPPs) and the formation of business clusters within the Greater Central Appalachian Voltage Valley (GCAVV) can potentially revitalize distressed communities in the Appalachian region. It introduces the concept of Clean Innovation Ecosystems (CIEs) as networks of organizations, institutions, and entrepreneurs working together to promote sustainable technologies, emphasizing the importance of government investment in these ecosystems to maximize social benefits and economic development in the region. Detailed Review/Feedback 1. Background of case’s history and progression Answer: Partly Feedback: The background provided in the paper is conceptually rich but lacks specific historical context and progression details regarding the Appalachian region's economic struggles and the evolution of the GCAVV. While the paper introduces the idea of Voltage Valleys and CIEs effectively, it does not sufficiently explain the historical development of these concepts or the specific challenges faced by the region. The authors should consider providing a more detailed historical overview, including key events, policies, and economic conditions that have shaped the region's current state, which could help readers better understand the context and significance of the proposed solutions. 2. Clarity and accuracy of presentation and citation of current literature Answer: No Feedback: The paper's presentation is ambitious, but it struggles with clarity and coherence. The integration of concepts like CIEs, business clusters, and public-private partnerships is not consistently clear, leading to confusion regarding the main arguments. The paper cites a range of sources, but many references are either outdated or not directly relevant to the specific case of the Appalachian region. To improve the paper, the authors should refine their focus, clarify key concepts, and ensure the literature cited is current and directly supports their arguments. A more structured presentation of ideas, possibly with clear subheadings and a logical flow of information, would substantially enhance the paper’s readability. 3. Statistical analysis and interpretation Answer: Not applicable Feedback: As there is no statistical analysis in the paper, this question is not applicable. However, the authors should consider including quantitative data or analysis to support their claims, especially when discussing the economic impacts of the proposed business clusters, such as data on job creation, investment levels, or economic growth in similar regions. 4. Availability of source data for reproducibility: Answer: Partly Feedback: The paper includes references to various sources and artifacts, but the reproducibility of the results is questionable due to the lack of detailed data. The supplementary materials are mentioned but not thoroughly integrated into the main text. To improve reproducibility, the authors should provide clearer links to the data sources, including any datasets or documents used in their analysis. They should also describe their data collection and analysis methods in more detail, enabling other researchers to replicate their study. 5. Support for conclusions Answer: No Feedback: The conclusions drawn in the paper are ambitious but not adequately supported by the evidence presented. The paper makes broad claims about the potential impact of CIEs and Voltage Valleys on distressed communities but does not provide sufficient empirical data or detailed case studies to substantiate these claims. The authors should strengthen their conclusions by presenting more concrete evidence, such as case studies of similar initiatives in other regions or quantitative data showing the impact of these ecosystems on economic development. Additionally, a more cautious and nuanced interpretation of the findings would make the conclusions more credible. 6. Usefulness of the case for teaching or other practitioners Answer: No Feedback: While the paper introduces interesting concepts, its practical applicability is limited by the lack of specific examples and actionable insights. The case is presented in a theoretical manner, which may not be immediately useful for practitioners or educators looking for real-world applications. To make the paper more useful for teaching or practice, the authors should include detailed case studies or examples of how CIEs and Voltage Valleys have been successfully implemented in other regions. They should also provide practical recommendations or guidelines for policymakers and practitioners interested in applying these concepts in similar contexts. Conclusion The paper presents an innovative and important topic with significant potential. However, it currently falls short in several key areas, including the clarity of its presentation, support for its conclusions, and practical applicability. To make the article scientifically sound and useful for a broader audience, the authors need to address the issues identified above.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/13-808
https://f1000research.com/articles/10-843/v1	23 Aug 21	{ "type": "Research Article", "title": "Hypoxic preconditioning promotes survival of human adipocyte mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers", "authors": [ "I Gde Rurus Suryawan", "Budi Susetyo Pikir", "Fedik Abdul Rantam", "Anudya Kartika Ratri", "Ricardo Adrian Nugraha", "Budi Susetyo Pikir", "Fedik Abdul Rantam", "Anudya Kartika Ratri", "Ricardo Adrian Nugraha" ], "abstract": "Background: Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study (in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression (p<0.001;β=-0.482) and hypoxia condition also influenced VEGF expression (p<0.001;β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression (p<0.001; β=0.985). The regression test results showed that time effects on HSP27 expression (p<0.001; β=0.398) and hypoxia precondition also affects HSP27 expression (p<0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis (p<0,001;β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.", "keywords": [ "apoptosis", "h-AMSCs", "BCL-2", "HSP27", "SCF", "VEGF expression" ], "content": "Abbreviation\n\nAMSC: Adipose Mesenchymal Stem Cells\n\nATP: Adenosine Triphosphate\n\nBAX: BCL-2-associated X protein\n\nBCL2: B-Cell Lymphoma 2\n\nCD44: Cluster of Differentiation 44\n\nh-AMSC: human Adipose Mesenchymal Stem Cells\n\nHSF1: Heat Shock Factor 1\n\nHSP27: Heat Shock Protein 27\n\nITD: Institute of Tropical Diseases (Universitas Airlangga)\n\nMANOVA: Multivariate Analysis of Variance\n\nOCT4: Octamer-binding transcription factor 4\n\nPK2: Protein k-2\n\nPKC: Protein kinase C\n\nROS: Reactive oxygen species\n\nSCF: Stem Cell Factor\n\nSLF: Steel Factor\n\nSPSS: Statistical Package for Social Sciences\n\nVEGF: Vascular Endothelial Growth Factor\n\n\nIntroduction\n\nSeveral literatures provide abundant information that human adipocytes mesenchymal stem cells (h-AMSCs) is an attractive resource for therapeutics and have beneficial effects in regenerating injured cardiomyocytes due to their self-renewal ability and broad differentiation potential under physiological and pathological conditions.1–3\n\nDespite the impressive potential of the h-AMSC-based therapy, several obstacles (e.g., the difficulty of maintaining self-renewal and poor survival due to apoptosis and/or necrosis at the administration site) have been encountered.4 Some studies suggest that more than 90% of transplanted stem cells, either intravenously, intramyocardially, and intracoronary delivery, have necrosis and apoptosis and only about 5% transplanted stem cells can survive up to 14 days in infarcted myocardium.5 The survival of stem cells transplantation is so poor because high percentage of dead cells due to factors such as limited availability of blood, hypoxia, oxidative stress, inflammatory processes, loss of extracellular cell buffer matrix (anoic), non-conducive microenvironment to myocardial infarction, structural damage to blood vessels and lack of nutritional support.6\n\nTherefore a particular strategy is needed to improve survival, increase proliferation, migration, maintain the potential for differentiation and viability of stem cells in environments with low oxygen levels. One of those strategies is to pre-condition hypoxic precursors in vitro on oxygen concentrations mimicking the stem cells’ niche.7,8 Contributing factors for improved survival of h-AMSCs cultured through hypoxia preconditioning, i.e., apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44 + expression.9\n\nIn detail, it has never been explained how far the role of hypoxic preconditions in inhibiting apoptosis of h-AMSCs culture in vitro, in order to enhance survival and increase proliferation, maintain multi-potency, stemness and inhibition of apoptosis. Based on the description above, we consider it is necessary to conduct a research to explain the increased survival of h-AMSCs through the treatment of sub-lethal hypoxia precondition (oxygen concentration of 1%) for 24, 48, and 72 hours by looking at the expression of inhibition on apoptosis and HSP27 expression, and BCL2. In addition, it is necessary to observe the role of hypoxic preconditions in the proliferation process through the expression of SCF, OCT-4, and BCL2.\n\nA study was conducted to explain and confirm the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs so that it could be used as a benchmark for h-AMSCs culture strategy before transplantation. This study was an experimental laboratory explorative study (in vitro study) with hypoxic preconditioning in human-adipose mesenchymal stem cells (h-AMSCs) cultures.\n\n\nMethods\n\nThe use of human subjects in this study had been obtaining an ethical approval from research ethics committee of Dr. Soetomo Academic General Hospital - Faculty of Medicine, Airlangga University (Number: 264/Panke.KKE/IV/2017) issued on April 6th, 2017 under the name of I Gde Rurus Suryawan as principal investigator.\n\nThis study is an exploratory laboratory experimental study (in-vitro study) with hypoxic preconditions in the culture of human-adipose mesenchymal stem cells (h-AMSCs) derived from human adipose tissue. The aim of this study was to obtain stem cells that have high survival so that they are not only viable but also have high adaptability to the environment when the stem cells are transplanted. This type of experiment is a true experimental post-test only control group design accompanied by phenotypic h-AMSCs characterization against CD44+, CD90 + and CD45- before being given treatment.\n\nThis research was conducted at the Center for Research and Development of Stem Cell - Institute Tropical Disease (ITD) Universitas Airlangga, Dr. Soetomo Academic General Hospital and the Faculty of Medicine, Airlangga University, Surabaya. The implementation of this study lasted for 2-3 months.\n\nThe sample size in this study was obtained using the Federer’s formula for sample size.10 This formula is used as a control for the degree of freedom in MANOVA. The formula description is as follows:\n\nSample size: (r-1) (K-1) ≥ 15\n\nr = replication (experimental unit sample size per group)\n\nK = number of subject group observations\n\nK = 6\n\n(r-1) (K-1) ≥ 15\n\n(r-1) (6-1) ≥ 15\n\n(r-1) 5 ≥ 15\n\nr-1 ≥ 3\n\nr = 4\n\nThen the number of replications for each group is 4, so that the total sample is 24 plate culture.\n\nExperimental Unit:\n\n1. h-AMSCs, namely human-adipose mesenchymal stem cells from adipose tissue obtained from minimally invasive surgery with small incisions (3-5 cm) under local anaesthesia in the lower abdominal area by a surgeon (Figure 1). These materials came from patients who were prepared for clinical application of stem cell therapy at the Network Bank Dr. Soetomo General Hospital, Surabaya. All procedures were approved by the relevant ethics committees, and written informed consent was obtained from all study participants. The h-AMSCs experimental unit was taken from adult patients who were in a stable state who were not taking anti-platelets or anti-coagulants and then multiplied in vitro at the 5th passage to 24 units. A total of 24 units were divided into two groups, namely control and treatment. The control group (P0) had 12 culture units in normoxic conditions (21% O2 concentration). The treatment group (P1) was 12 units pre-conditioned to hypoxia (1% O2 concentration). Both treatment groups were observed for survival (CD44+, VEGF, SCF, OCT-4, BCL2, HSP27, and apoptotic inhibition at 24, 48 and 72 hours of cell culture). Observation of apoptotic inhibition based on the expression of BCL2 and HSP27 along with the percentage of apoptosis that occurred.\n\n2. Washing buffer (phosphate-buffered saline, PBS, Sigma-Aldrich, Milan, Italy, 0.1% sodium azide, and 0.5% bovine serum albumin (BSA), Radnor, USA) was used for all washing steps (3 ml of washing buffer and centrifugation, 400g for eight minutes at 4°C). Briefly, 5 × 105 cells/sample were incubated with 100 ml of 20 mM ethylene-diaminetetraacetic acid (EDTA, Sigma-Aldrich) at 37°C for 10 minutes and washed.\n\nIsolation and culture of h-AMSCs from the patient's adipose tissue (human).\n\nThis research was conducted in four stages as follows:\n\n1. Isolation and culture of h-AMSCs from the patient's adipose tissue (human) (Figure 1).\n\n2. Characterization of h-AMSCs from adipose tissue phenotypically (Flowcytometry) through identification of CD44+, CD90+ and CD45- before being treated with hypoxic preconditions.\n\n3. Hypoxic precondition in in vitro h-AMSCs culture was carried out with an oxygen concentration of 1% for 24, 48 and 72 hours.\n\n4. Observation of survival of h-AMSCs in the form of CD44 +, VEGF, SCF, OCT-4, BCL2, HSP27 expression, and apoptotic inhibition:\n\na. Phenotype expression of CD44 + was carried out by the flowcytometric method.\n\nb. Immuno-cytochemical expression of VEGF\n\nc. Immunocytochemical expression of SCF from h-AMSCs culture\n\nd. Phenotype of OCT-4 expression (Immunocytochemistry and Immunofluorescence)\n\ne. Apoptotic inhibition, based on the expression of BCL2 and HSP27 by immunocytochemistry accompanied by a low percentage of apoptosis through the Tunnel Assay method (Figure 2).\n\nObservation of survival of h-AMSCs in the form of CD44 +, VEGF, SCF, OCT-4, BCL2, HSP27 expression, and apoptotic inhibition:\n\nA. Phenotype expression of CD44 + was carried out by the flowcytometric method.\n\nB. Immuno-cytochemical expression of VEGF.\n\nC. Immunocytochemical expression of SCF from h-AMSCs culture.\n\nD. Phenotype of OCT-4 expression (Immunocytochemistry and Immunofluorescence).\n\nData collected, processed and statistically tested with several stages. The first stage is an Assumption Test in the form of a normality test to ensure that the data is normally distributed. Furthermore, a comparison test was carried out between the treatment group and the control group using Multivariate Analysis of Variance (MANOVA). Furthermore, path analysis is carried out to determine the pathway mechanism of the influence of the independent variables on the dependent variable by using multiple linear regression statistical tests. The statistical analysis was used to explain the effect of time (24, 48 and 72 hours) and hypoxic conditions on the expression of VEGF, SCF, OCT-4, CD44 +, BCL2, HSP27 and the number of cells undergoing apoptosis. The data scale of each variable under study is a ratio, so it is appropriate to use the Multiple Linear Regression statistical test. Statistical tests were performed using SPSS version 24.0 software.\n\n\nResults\n\nThe results showed that the time difference test on CD44+ expression was 24 hours with 48 hours (p = 0.017), 24 hours with 72 hours (p = 0.004), and 48 hours with 72 hours (p = 0.801). The result of regression test showed that time difference had an effect on expression of CD44 + (p = 0.002, β = −0.582) and hypoxia condition had no effect to CD44 + expression (p = 0.066, β = 0,317) (Table 1) (Figure 3).\n\nResults on CD44+ expression.\n\nFlowcytometry results from human AMSCs based on cell culture for CD44+ CD90+ CD45- expression.\n\nThe result of time difference test on VEGF expression is between 24 hours with 48 hours (p < 0.001), 24 hours with 72 hours (p < 0.001), and 48 hours with 72 hours (p=0.047). The result of regression test showed that time difference had an effect on VEGF expression (p < 0.001; β =−0,482) and hypoxia condition also influenced VEGF expression (p < 0.001; β = 0,774) (Table 2) (Figure 4).\n\nResults on VEGF expression.\n\nImmunohistochemical Characteristic of h-AMSCs based on VEGF expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.\n\nThe result of time difference test on SCF expression is between 24 hours with 48 hours (p = 0.283), 24 hours with 72 hours (p < 0.001), and 48 hours with 72 hours (p < 0.001). The result of path analysis showed that VEGF influenced the expression of SCF (p < 0.001; β = 0.889) (Table 3) (Figure 5).\n\nResults on SCF expression.\n\nImmunohistochemical Characteristic of h-AMSCs based on SCF expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.\n\nThe result of time difference test on OCT-4 expression is between 24 hours with 48 hours (p < 0.001), 24 hours with 72 hours (p < 0.001), and 48 hours with 72 hours (p < 0.001). The result of path analysis showed that SCF had an effect on OCT-4 expression (p < 0.001; β = 0.985) (Table 4) (Figure 6).\n\nResults on OCT4 expression.\n\nImmunohistochemical Characteristic of h-AMSCs based on OCT-4 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours. Immunofluorescence assay of h-AMSCs based on OCT-4 expression at: G) normoxic condition for 24 hours; H) normoxic condition for 48 hours; I) normoxic condition for 72 hours; J) hypoxic condition for 24 hours; K) hypoxic condition for 48 hours; L) hypoxic condition for 72 hours.\n\nThe results of time difference test on BCL2 expression between 24 hours with 48 hours (p = 0.223), 24 hours with 72 hours (p = 0.295), and 48 hours with 72 hours (p = 0.982). Path analysis results show that OCT-4 effect on BCL2 expression (p < 0.001; β = 0.878) (Table 5) (Figure 7).\n\nResults on BCL2 expression.\n\nImmunohistochemical Characteristic of h-AMSCs based on BCL2 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.\n\nThe results of the time difference test on HSP27 expression between 24 hours with 48 hours (p = 0.040), 24 hours with 72 hours (p < 0.001), and 48 hours with 72 hours (p < 0.001). The regression test results showed that time effects on HSP27 expression (p < 0.001; β = −0.398) and hypoxia precondition also affects HSP27 expression (p < 0.001; β = 0.847) (Table 6) (Figure 8).\n\nResults on HSP27 expression.\n\nImmunohistochemical Characteristic of h-AMSCs based on HSP27 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.\n\nThe results of time difference test on number of apoptotic cell amount between 24 hours with 48 hours (p = 0.004), 24 hours with 72 hours (p = 0.562), and 48 hours with 72 hours (p < 0.001). Pathway analysis showed that BCL2 expression inhibited apoptosis (p = 0.030; β =−0.442) and HSP27 expression also inhibited apoptosis (p < 0.001; β =−0.487) (Table 7) (Figure 9).\n\nResults on number of apoptotic cell amount.\n\nImmunohistochemical Characteristic of h-AMSCs based on number of apoptotic cell amount at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.\n\n\nDiscussion\n\nOver the last few years, with the gradual increase in awareness of the critical role that hypoxia-induced signalling could play as a tool for generating angiogenesis on demand, two distinct approaches have emerged, as promising strategies to achieve this goal.5 On one hand, researchers have explored the possibility of pre-conditioning cells or grafts to hypoxia in vitro, in order to upregulate the required signalling that can then initiate angiogenesis in vivo upon transplantation.11 The second approach relies on direct induction of hypoxia-mediated signalling in vivo, by pharmacological means or gene therapy.12 A further distinction can be made on whether the therapy involves transplantation of hypoxia pre-conditioned or genetically modified cells, or if the effect is mediated directly through gene transfer or cell-free delivery of hypoxia-induced protein factors.13\n\nThe low survival of h-AMSCs after transplanting the heart muscle with myocardial infarction has limited the effectiveness of stem cell therapy.8 This is presumably because the transplanted stem cells are difficult to adapt to a new environment that is different from the environment during the in vitro culture process if it is carried out under normoxic conditions (21% oxygen concentration), while the niche of h-AMSCs in adipose tissue is actually under hypoxic conditions (oxygen concentration between 2-8%).14 The mechanism underlying the decreased effectiveness of stem cells when transplanted is thought to be because many transplanted stem cells undergo apoptosis.15 Therefore, a strategy is needed to increase the resistance of transplanted stem cells, one of which is the hypoxic adaptation process during in vitro culture.16\n\nThe role of sub lethal hypoxia during the in vitro culture process is to provide hypoxic preconditions so that the support niche is compatible with the hypoxic environment in vivo in myocardial infarction.17 Hypoxic precondition will trigger Vascular Endothelial Growth Factor (VEGF) which then binds to VEGF Receptor-1 (VEGFR-1) in the cytosol.18 The presence of VEGF - VEGFR-1 bonds is thought to occur in a series of signalling which activates Stem Cell Factor (SCF) or Steel Factor (SLF) in the interstitial.19 Interstitial SCF expression will be recognized by the SCF receptor so that an SCF-receptor complex is formed in the cell nucleus and nuclear β1-integrine expression will activate Octamer-4 (OCT-4) so that stem cells experience proliferation, self-renewal but still have the potential for differentiation.20,21 OCT-4 also plays a role in the activation of the PI3/Akt pathway which affects survival cells by increasing BCL2 in the cytosol, resulting in inhibition of BAX, which causes mitochondrial PT-Pore to remain closed.22 The closure of the PT-Pore from the mitochondria will inhibit the release of Cytochrome-C and Apoptotic protease activating factor-1 (APAF-1) so that the apoptotic cascade does not occur.23\n\nFurthermore, the hypoxic precondition will lead to the expression of Cluster of Differentiation 44+ (CD44+).24 This CD44+ expression occurs due to stimulation of the nuclear β1 integrin from the cell nucleus which is expressed due to the presence of the SCF-receptor complex bond.25 CD44+ is a hyaluronan receptor which is part of the adhesion molecule, causing interactions between cells and between cells and the matrix, as well as lymphocyte activation, also plays a role in the homing process, and increases cell migration.26 CD44+ is a polymorphic family that is immunologically related to proteoglycans and cell surface glycoproteins as markers of h-AMSCs. Apart from being a marker for h-AMSCs, CD44 + has a signalling function that plays a role in cell survival and motility.26\n\nOn the other hand, hypoxic conditions are thought to have an effect on mitochondria in increasing the expression of Reactive Oxygen Species (ROS).27 The increased ROS due to hypoxic conditions is thought to be the cause of the increase in free radicals formed through mitochondrial-mediated pathways.28 This triggers protein kinase-C (PKC) and protein K-2 (PK2) which then triggers the p53 gene so that there is an increase in p53 protein expression which will activate proapoptotic members such as BAX.29 Increased expression of p53 causes mitochondrial damage which causes pores to open in the membrane, so that Cytochrome-C and other molecules that act as APAF-1 will exit the mitochondria.30 This condition will activate procaspase 9 to become caspase-9 and followed by activation of procaspase 3 to become active caspase-3 which affects DNase so that DNA fragmentation occurs, and ends with cell death through the apoptosis process.29\n\nHowever, the low sublethal oxygen concentration is thought to activate cells for protection in the form of repair.31 The repair process can be done through the activation of heat shock factor-1 (HSF-1) so that the formation of several Heat Shock Proteins (HSPs) occurs.32 HSPs are the product of several gene families contained in the cell nucleus which act as chaperone molecules that play a role in cell survival during the stress process.33 Some of the HSPs that were thought to be involved were HSP70, HSP90α and HSP27.32 However, in hypoxic conditions that cause the glycolysis process. This glycolysis process will further affect Krebs’s cycles so that ATP synthesis decreases.34 This decrease in ATP concentration is thought to cause a decrease in the function of HSP70 and HSP90α. This is because HSP70 and HSP90α are ATP-dependent chaperone molecules, thus the two HSPs (HSP70 and HSP90α) do not have the ability to act as chaperones in protecting, protecting and repairing cells under stress.35 The role of chaperone molecules in hypoxic conditions is carried out by HSP27, because HSP27 is ATP-independent chaperone. In addition, hypoxic precondition can maintain multipotential properties through OCT-4 expression compared to normoxic conditions.36\n\n\nConclusion\n\nFrom this study, it can be concluded that the hypoxic preconditioning affect the survival of h-AMSC with different apoptotic presentations due to the increased expression of BCL2 (anti apoptotic protein) and HSP 27 as chaperone proteins that play a role in inhibiting apoptosis. In this study, the hypoxic preconditioning may elevate the expression of studied variables, such as the number of apoptosis through BCL2 and HSP27 expression, trigger signal through VEGF expression, proliferation through SCF expression, and multipotency through OCT-4 expression. Hypoxic preconditioning significantly affects VEGF, VEGF affects SCF expression, SCF affects OCT-4 expression, OCT-4 affects BCL2 expression, but hypoxia also affects HSP27 expression. BCL2 and HSP27 have proven inhibiting apoptosis thus enhancing h-AMSCs survival (Figure 10). In conclusion, hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27. But CD 44+ did not play a role in the mechanism of survival improvement of human AMSC survival.\n\nPath analysis with MANOVA and multiple linear regression analysis for hypoxic preconditioning in h-AMSCs survive.\n\n\nData availability\n\nFigshare: Raw Data - Hypoxic Preconditioning Promotes Survivals of Human Adipocyte Mesenchymal Stem Cell via Expression of Prosurvival and Proangiogenic Biomarkers. https://doi.org/10.6084/m9.figshare.15029016.v1.37\n\nThis project contains the following underlying data:\n\n• Data file 1. Raw Data.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nWe would also like to show our gratitude to Dr. Andrianto and Dr. Meity Ardiana for sharing their pearls of wisdom with us during the writing process, and we thank for anonymous residents and staffs for their so-called insights. We are also immensely grateful to all professors and consultants from Department of Cardiology and Vascular Medicine – Faculty of Medicine, Universitas Airlangga for their comments on an earlier version of the manuscript, although any errors are our own and should not tarnish the reputations of these esteemed persons.\n\nA previous version of this paper is available at bioRxiv 2021.01.18.427057; https://www.biorxiv.org/content/10.1101/2021.01.18.427057v1).\n\n\nReferences\n\nStępniewski J, Tomczyk M, Andrysiak K, et al.: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, in Contrast to Adipose Tissue-Derived Stromal Cells, Efficiently Improve Heart Function in Murine Model of Myocardial Infarction. Biomedicines. 2020; 8(12): 1–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAcquistapace A, Bru T, Lesault PF, et al.: Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer. Stem Cells. 2011; 29(5): 812–824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee J, Lee S, Lee CY, et al.: Adipose-derived stem cell-released osteoprotegerin protects cardiomyocytes from reactive oxygen species-induced cell death. Stem Cell Res Ther. 2017; 8(1): 4–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBagheri-Hosseinabadi Z, Mesbah-Namin SA, Salehinejad P, et al.: Fibrin scaffold could promote survival of the human adipose-derived stem cells during differentiation into cardiomyocyte-like cells. Cell Tissue Res. 2018; 372(3): 571–589. PubMed Abstract \| Publisher Full Text\n\nLauvrud AT, Kelk P, Wiberg M, et al.: Characterization of human adipose tissue-derived stem cells with enhanced angiogenic and adipogenic properties. J Tissue Eng Regen Med. 2017; 11(9): 2490–2502. PubMed Abstract \| Publisher Full Text\n\nGreen LJ, Zhou H, Padmanabhan V, et al.: Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles. Stem Cell Res Ther. 2019; 10(1): 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYue Y, Zhang P, Liu D, et al.: Hypoxia preconditioning enhances the viability of ADSCs to increase the survival rate of ischemic skin flaps in rats. Aesthetic Plast Surg. 2013; 37(1): 159–170. PubMed Abstract \| Publisher Full Text\n\nWang JW, Qiu YR, Fu Y, et al.: Transplantation with hypoxia-preconditioned mesenchymal stem cells suppresses brain injury caused by cardiac arrest–induced global cerebral ischemia in rats. J Neurosci Res. 2017; 95(10): 2059–2070. PubMed Abstract \| Publisher Full Text\n\nWang XY, Liu CL, Li SD, et al.: Hypoxia precondition promotes adipose-derived mesenchymal stem cells based repair of diabetic erectile dysfunction via augmenting angiogenesis and neuroprotection. PLoS One. 2015; 10(3): 1–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoordzij M, Dekker FW, Zoccali C, et al.: Sample size calculations. Nephron - Clin Pract. 2011; 118(4): 319–323. PubMed Abstract \| Publisher Full Text\n\nNouri F, Nematollahi-Mahani SN, Sharifi AM: Preconditioning of mesenchymal stem cells with non-toxic concentration of hydrogen peroxide against oxidative stress induced cell death: The role of hypoxia-inducible factor-1. Adv Pharm Bull. 2019; 9(1): 76–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu C, Li L: Preconditioning influences mesenchymal stem cell properties in vitro and in vivo. J Cell Mol Med. 2018; 22(3): 1428–1442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHou J, Wang L, Long H, et al.: Hypoxia preconditioning promotes cardiac stem cell survival and cardiogenic differentiation in vitro involving activation of the HIF-1α/apelin/APJ axis. Stem Cell Res Ther. 2017; 8(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMas-Bargues C, Sanz-Ros J, Román-Domínguez A, et al.: Relevance of oxygen concentration in stem cell culture for regenerative medicine. Int J Mol Sci. 2019; 20(5). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu H, Xue W, Ge G, et al.: Hypoxic preconditioning advances CXCR4 and CXCR7 expression by activating HIF-1α in MSCs. Biochem Biophys Res Commun. 2010; 401(4): 509–515. PubMed Abstract \| Publisher Full Text\n\nTheus MH, Wei L, Cui L, et al.: In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain. Exp Neurol. 2008; 210(2): 656–670. PubMed Abstract \| Publisher Full Text\n\nWei ZZ, Zhu YB, Zhang JY, et al.: Priming of the cells: Hypoxic preconditioning for stem cell therapy. Chin Med J (Engl). 2017; 130(19): 2361–2374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamakrishnan S, Anand V, Roy S: Vascular endothelial growth factor signaling in hypoxia and inflammation. J Neuroimmune Pharmacol. 2014; 9(2): 142–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakayama: Erratum: Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro (Blood (April 1, 2000) 95 (2275-2283)). Blood. 2000; 96(4): 1273. Publisher Full Text\n\nGilmore AP, Metcalfe AD, Romer LH, et al.: Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J Cell Biol. 2000; 149(2): 431–445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBachelder RE, Ribick MJ, Marchetti A, et al.: p53 Inhibits α6β4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol. 1999; 147(5): 1063–1072. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernardi P, Di Lisa F: The mitochondrial permeability transition pore: Molecular nature and role as a target in cardioprotection. J Mol Cell Cardiol. 2015; 78: 100–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorsmeyer SJ, Wei MC, Saito M, et al.: Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 2000; 7(12): 1166–1173. PubMed Abstract \| Publisher Full Text\n\nLiang G, Li S, Du W, et al.: Hypoxia regulates CD44 expression via hypoxia-inducible factor-1α in human gastric cancer cells. Oncol Lett. 2017; 13(2): 967–972. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun Z, Guo SS, Fässler R: Integrin-mediated mechanotransduction.2016; 215(4).\n\nMisra S, Hascall VC, Markwald RR, et al.: Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer. Front Immunol. 2015; 6(MAY). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSena LA, Chandel NS: Physiological roles of mitochondrial reactive oxygen species. Mol Cell. 2012; 48(2): 158–167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchieber M, Chandel NS: ROS function in redox signaling. Curr Biol. 2014; 24(10): 453–462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVolpe CMO, Villar-Delfino PH, Dos Anjos PMF, et al.: Cellular death, reactive oxygen species (ROS) and diabetic complications review-Article. Cell Death Dis. 2018; 9(2). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarrido C, Galluzzi L, Brunet M, et al.: Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006; 13(9): 1423–1433. PubMed Abstract \| Publisher Full Text\n\nDarby IA, Hewitson TD: Hypoxia in tissue repair and fibrosis. Cell Tissue Res. 2016; 365(3): 553–562. PubMed Abstract \| Publisher Full Text\n\nJacob P, Hirt H, Bendahmane A: The heat-shock protein/chaperone network and multiple stress resistance. Plant Biotechnol J. 2017; 15(4): 405–414. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShende P, Bhandarkar S, Prabhakar B: Heat Shock Proteins and their Protective Roles in Stem Cell Biology. Stem Cell Rev Reports. 2019; 15(5): 637–651. PubMed Abstract \| Publisher Full Text\n\nRyall JG, Cliff T, Dalton S, et al.: Metabolic Reprogramming of Stem Cell Epigenetics. Cell Stem Cell. 2015; 17(6): 651–662. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Q, Li X, Wang Q, et al.: Heat shock pretreatment improves mesenchymal stem cell viability by heat shock proteins and autophagy to prevent cisplatin-induced granulosa cell apoptosis. Stem Cell Res Ther. 2019; 10(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernandez I, Baio JM, Tsay E, et al.: Short-term hypoxia improves early cardiac progenitor cell function in vitro. Am J Stem Cells. 2018; 7(1): 1–17. Reference Source\n\nSuryawan IGR, Pikir BS, Rantam FA, et al.: Raw Data - Hypoxic Preconditioning Promotes Survivals of Human Adipocyte Mesenchymal Stem Cell via Expression of Prosurvival and Proangiogenic Biomarkers. Figshare. 2021." }	[ { "id": "128761", "date": "27 Apr 2022", "name": "Gun-Jae Jeong", "expertise": [ "Reviewer Expertise Mesenchmal stem cells", "tissue engineering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 entitled “Hypoxic preconditioning promotes survival of human adipocyte mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers” by I Gde Rurus Suryawan et al. (2021) reported enhanced survival of human adipocyte mesenchymal stem cell with hypoxic preconditioning. This study is well designed, and several experiments are performed well. However, this manuscript needs to be modified in certain minor aspects:\nMinor comments:\nPlease revise the adipocyte mesenchymal stem cell as adipose derived mesenchymal stem cell or other appropriate word. The word adipocyte already contains the meaning of cell (-cyte). Therefore, the meanings are overlapped in a word.\n\nPlease provide more information of immunocytochemistry methods and antibody information in materials and methods section.\n\nPlease provide detailed quantification methods for each quantification. What is the measurement of each quantification experiment?\n\nOverall figures: please include scale bars for each figure set. It looks like they have different magnifications.\n\nOverall figures: please resize each subset figures as same size in one 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8158", "date": "19 Oct 2022", "name": "Ricardo Adrian Nugraha", "role": "Author Response", "response": "Dear Prof. Gun Jae Jeong Thank you for your comment and suggestion. We are happy to get valuable comment regarding our work. Author Response Letter to Reviewer Comments: Please revise the adipocyte mesenchymal stem cell as adipose derived mesenchymal stem cell or other appropriate word. The word adipocyte already contains the meaning of cell (-cyte). Therefore, the meanings are overlapped in a word. -> We will revised as your suggestion. Please provide more information of immunocytochemistry methods and antibody information in materials and methods section. -> We will provide more information regarding the immunocytochemistry methods and antibody information Please provide detailed quantification methods for each quantification. What is the measurement of each quantification experiment? -> We will provide detailed quantification methods Overall figures: please include scale bars for each figure set. It looks like they have different magnifications. -> Unfortunately, scale bars is difficult to achieve since we got it from the third party Overall figures: please resize each subset figures as same size in one figure. -> We will resize our figure in the same size" }, { "c_id": "8619", "date": "19 Oct 2022", "name": "Ricardo Adrian Nugraha", "role": "Author Response", "response": "The article entitled “Hypoxic preconditioning promotes survival of human adipocyte mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers” by I Gde Rurus Suryawan et al. (2021) reported enhanced survival of human adipocyte mesenchymal stem cell with hypoxic preconditioning. This study is well designed, and several experiments are performed well. However, this manuscript needs to be modified in certain minor aspects: Minor comments: Please revise the adipocyte mesenchymal stem cell as adipose derived mesenchymal stem cell or other appropriate word. The word adipocyte already contains the meaning of cell (-cyte). Therefore, the meanings are overlapped in a word. Author Response: Thank you for your suggestion, author have already replace adipocyte mesenchymal stem cell with adipose derived mesenchymal stem cell Please provide more information of immunocytochemistry methods and antibody information in materials and methods section. Author Response: Thank you for your suggestion. We have provided additional information regarding the immunocytochemistry methods and antibody information Please provide detailed quantification methods for each quantification. What is the measurement of each quantification experiment? Author Response: We measured mean fluorescence intensity (MFI) in a region of interest (ROI) with ImageJ software. We were simply measuring automatic cell counting based on the total fluorescent intensity across the entire image that lead to the potentially erroneous conclusion that the sample with the smaller area/fewer cells exhibits less staining for the probe of interest than one that is larger in area or has more cells, when the reality is that they actually exhibit similar staining levels per tissue area Overall figures: please include scale bars for each figure set. It looks like they have different magnifications. Author Response: Figure 3-10 had similar magnification. Unfortunately, figure 1 and 2 had different magnification. We try to add scale bare but it couldn’t work well. Overall figures: please resize each subset figures as same size in one figure. Author Response: Figure 3-10 had been resize as same size in one figure. Unfortunately, we try to resize figure 1 and 2 but it couldn’t be better." } ] } ]	1	https://f1000research.com/articles/10-843
https://f1000research.com/articles/13-803/v1	16 Jul 24	{ "type": "Research Article", "title": "Rorschach and resting state-fMRI: Personality Assessment and unique associations with resting-state brain networks activity", "authors": [ "Stefania Cristofanelli", "Enrico Vitolo", "Alessandro Zennaro", "Franco Cauda", "Tommaso Brischetto Costa", "Eleonora Centonze", "Giorgia Baccini", "Jordi Manuello", "Laura Ferro", "Alessandro Zennaro", "Franco Cauda", "Tommaso Brischetto Costa", "Eleonora Centonze", "Giorgia Baccini", "Jordi Manuello", "Laura Ferro" ], "abstract": "Background The Rorschach test is one of the most popular tests used in clinical settings for psychopathology and personality assessment; however, there is still little evidence regarding the functional correlates of test responses. Functional magnetic resonance imaging (fMRI) allows for the investigation of biological correlates associated with different psychological functions. Among other applications, fMRI has been used to identify different networks that reflect brain activity in the resting state (rs-fMRI), that is, when an individual is not engaged in any specific task. Among these resting-state networks (RSN), the best-known and most studied are the Default Mode Network (DMN), along with other networks such as salience, frontoparietal, sensorimotor, temporo-parietal, visual, and cerebellar networks.\n\nMethods We used this type of analysis to obtain new evidence regarding Rorschach. This study aimed to analyze the functional brain architecture underlying Rorschach’s personality variables, administered according to Exner’s Comprehensive System (CS). In particular, the aims are: 1) to ascertain the existence of correlations between CS variables and activity of the RSN, and 2) to use these profiles of activity to develop a new data-driven clustering of the CS variables. Archival data from twenty-four non-clinical subjects were analyzed. Independent Component Analysis (ICA) and partial least squares regression (PLS) were used to analyze the fMRI data.\n\nResults The results showed specific associations with the given Rorschach variables (several of which could be grouped into higher-order latent factors) and activity of the main RSN. Moreover, the cluster analysis outlined important groupings of Rorschach variables, particularly regarding their clinical implications.\n\nConclusions Our study could be comprised in the existing literature providing strong evidence about the neurobiological validity of the Rorschach test.", "keywords": [ "Rorschach", "Comprehensive System", "Neuroimaging", "fMRI", "resting-state networks." ], "content": "1. Introduction\n\nThe Rorschach Comprehensive System (CS) is a standardized performance-based test that shows in vivo how subjects combine attention, perception, and logical analysis to cope with a task. These strategies are also applied in daily life and can be considered as the expression of the personality “in action.”\n\nSpecifically, the test provides information on both structure and dynamic functioning of personality; the term “structure” indicates the combination of personality states (temporary and situation-dependent set of characteristics, attitudes and moods) and traits (stable characteristics and attitudes). The concept of “functioning” describes how states and traits guide one’s perception of the world and behavior, consistent with one’s own beliefs and needs (Exner, 2003; Exner & Erdberg, 2005).\n\nThe test consisted of 10 cards, each showing an inkblot for which the subject was asked to answer the question “What could it be?” (Exner, 2003). The goal was to detect personality features through the answers provided in the test. The purpose, therefore, is to observe what the person “is”, instead of what the person “says he is”. In other words, explicit behaviors measured by the test are assumed to reliably reflect the implicit dimensions of psychological functioning.\n\nIn 1970, J. E. Exner began a systematic investigation to make Rorschach method a test: standardize and objective the Rorschach method. The first version of the Comprehensive System was published in 1974, and over the years, it will undergo numerous revisions, the last of which dates back to 2005. Exner, following Rorschach’s thought, emphasizes the role of perceptual processes, which are clearly distinct from associative ones. A projective approach considers the content of a response as a product of the unconscious. According to Exner, projection plays a core role only in some phases of the response process: at the beginning, when the stimulus is “scanned,” and at the end, when the subject personalizes the answer. In all other phases, cognitive and perceptual mechanisms, such as attention or the processing of inkblots, predominate (Exner, 2003; Exner & Erdberg, 2005). After the answer phase, there is an inquiry that aims to investigate the response process to understand how the subject got to the answer (Exner, 2003). Finally, the collected data are transformed into a series of indices reported in the Structural Summary (SS) (Exner & Erdberg, 2005).\n\nExner’s work has provided instruments with good psychometric properties. In particular, the test has a high degree of inter-rater reliability; excellent test-retest fidelity; and adequate convergent, divergent, and predictive validity (Exner, 2003; Exner & Erdberg, 2005).\n\nMost of the brain’s energy is directed to support its activity at rest (Raichle, 2011). Spontaneous activity that occurs in the brain when it is not engaged in any specific task consumes more than 80% of the brain’s energy (Rosazza & Minati, 2011). These data suggest that spontaneous brain activity is associated with what is called “present state” or “mental state” (Raichle, 2011). In the last 20 years, resting-state functional Magnetic Resonance Imaging (rs-fMRI) has been used to investigate physiological correlates of spontaneous brain activity (Canli & Amin, 2002; Chen et al., 2020). The goal of neuroimaging studies that measure brain activity at rest is to identify spontaneous neuronal oscillations in large areas of the brain when the person is not engaged in any specific task (Buckner & Vincent, 2007). Furthermore, the tool allows us to study functional connectivity, that is, synchronous neural activation, among brain regions from an anatomical point of view, which may not even be close to each other (Rosazza & Minati, 2011).\n\nDespite the growing interest in large-scale resting-state brain networks, many aspects of their function (DeYoung et al., 2010). Nevertheless, there are several hypotheses, such as the idea that spontaneous activity reflects cognitive processes and that they reflect a combination of conscious activity and internal neural dynamics that could be essential for the emergence of behaviors, although they appear even in the absence of a behavioral correlate (Rosazza & Minati, 2011). However, two aspects are well known: 1) these patterns have a high test-retest reliability, which means that they are stable over time (DeYoung et al., 2010; Teeuw et al., 2021; Zuo et al., 2010); and 2) resting-state connectivity is strictly linked to one’s history and experience (Sporns, 2013). Therefore, it is reasonable to assume that brain activity during REST reflects individual differences in several healthy and pathological psychological variables.\n\nOver the years, several brain networks associated with functional connectivity during resting conditions have been identified. Despite methodological differences, the most supported across different studies are the following networks: the default mode network (DMN), sensorimotor network, executive control network, two lateralized frontoparietal networks (FPN), visual network, temporoparietal (auditory) network, dorsal attention network (DAN), and salience network (SN) (Lund et al., 2022; Razi et al., 2017; Rosazza & Minati, 2011).\n\nNowadays, the more studied more because it is more active when people are at rest than when they are engaged in a task (Rosazza & Minati, 2011), although the exact cognitive meaning of its activity is not yet fully clear (Mancuso et al., 2022). While it is generally described as a unitary network, three main sub-components can be identified, each possibly underlying a slightly different cognitive domain. The first encompasses the medial prefrontal cortex (mPFC), which is crucial for receiving sensory information coming both from the external world and from inside the body, which is then conveyed to structures such as the hypothalamus, amygdala, and periaqueductal gray (PAG). This pattern suggests a role of the DMN in the regulation of social behavior and mood and in motivational drive, three aspects that play an important role in shaping one’s personality (Raichle, 2015). The second sub-component consists of the dorsomedial prefrontal cortex (dmPFC) and is associated with self-referential judgments (Gusnard et al., 2001). The third sub-component includes the activation of the posterior cingulate cortex (PCC), adjacent precuneus, and lateral parietal cortex. The last module plays an important role in the evocation of previously formed memory. In fact, it is associated with hippocampal formation, a structure long known for its involvement in memory processes (Shannon et al., 2013; Yeshurun et al., 2021). The activity of the DMN has also been observed in non-human primates, which may suggest on the one hand that it is a stable component across various species and, on the other, that it is an intrinsic property of the brain (Rosazza & Minati, 2011).\n\nIf the DMN is the most studied component, the sensorimotor network will be the first to be identified using resting-state fMRI (Biswal et al., 1995, 2010). It complies with the pre-motor gyrus, post-motor gyrus, and supplementary motor areas. The overlap between this network and the sensory and motor components is not only anatomical but also functional; indeed, this network seems to be activated during the execution of (active) motor tasks. The question, therefore, is how the same pattern that is activated during the execution of a task is consistently activated in resting conditions. One hypothesis is that regions that are activated together during active tasks tend to be activated together during resting stages (a type of procedural neural memory (Rosazza & Minati, 2011).\n\nAnother resting-state network is the visual network, for which there is no agreement on the number and components of the network. The most solid findings are in line with defining this network as formed by mesial visual areas, that is, the striate cortex and typically mesial extra-striate regions (e.g., the lingual gyrus), and lateral visual areas (e.g., occipital pole and occipito-temporal regions). Stevens et al. (2010) observed that fluctuations in this component were modulated by a visual task that was completed before data acquisition. This result seems to support the hypothesis that resting states have experience-dependent dynamic components that may play a role in memory consolidation.\n\nThe executive control network comprises three components that are generally involved in tasks that require the intervention of executive functions, such as working memory tasks: the medial frontal gyrus, superior frontal gyrus, and anterior cingulate cortex (Rosazza & Minati, 2011; Seeley et al., 2007).\n\nThe FPN is defined by two highly lateralized components: one in the left hemisphere and the other in the right hemisphere. This circuit includes the inferior frontal gyrus, medial frontal gyrus, precuneus, inferior parietal lobule, and the angular gyrus. In addition, the brain areas implicated in this circuit represent the neural correlates of a plethora of cognitive processes ranging from memory to language (Rosazza & Minati, 2011).\n\nThe temporoparietal network is composed of the inferior frontal gyrus, medial and superior portions of the temporal gyrus, and the angular gyrus. The anatomical structures involved in this circuit are usually associated with language and, although the functions are not yet completely clear (Rosazza & Minati, 2011), it seems that the activation of this ls-rsbn network corresponds to the functional organization of language processes (Rosazza & Minati, 2011; Dronkers et al., 2004; Koyama et al., 2010; Turken & Dronkers, 2011).\n\nDAN is an important mediator of goal-directed and selective attentional processing. It includes the frontal eye fields, the posterior and anterior intra-parietal sulci, and the middle temporal visual area (Lanssens et al., 2020; Allan et al., 2019; Raichle, 2011; Razi et al., 2017).\n\nThe SN is involved in the interoception of feelings associated with gratification and consists of synchronous activation of the insula, anterior cingulate cortex, and ventral striatum (Pace-Schott & Picchioni, 2017). Its connection to other networks suggests that the SN also contributes to a wide variety of mental functions such as communication and social behavior (Menon, 2015). Although the results are not yet fully clear, some studies have shown activation of this network in response to an experience of social rejection (Redcay & Warnell, 2018).\n\nFinally, the last network is represented by the cerebellum, whose functions are not limited only to the motor field but also expand to cognitive processes (Guell et al., 2018; Tang et al., 2022).\n\nCognitive psychologists have used neuroimaging techniques to study brain connectivity underlying functions such as memory or language. The study of neurobiological correlates of complex psychological constructs, such as human personality or psychopathology, is complicated. Recently, however, a growing number of studies have investigated the ancestral neuronal roots of personality and the biological correlates of many psychiatric disorders (Panksepp & Davis, 2020).\n\nBased on the literature presented so far, it is possible to ask whether neuroimaging techniques can identify the neural correlates of personality, as measured by the Rorschach CS. Otherwise, can the Rorschach CS predict some neural parameters?\n\nCurrently, only a few studies have investigated the relationship between Rorschach responses and their neural correlates. For instance, Giromini and colleagues (2017b) found greater cortical activations of temporo-occipital and fronto-parietal areas in individuals that were looking at ten Rorschach cards compared to simple fixation cross conditions. Furthermore, specific associations between the activity of the mirror neuron system and human movement responses have been found among non-clinical subjects (Giromini, Viglione, Pineda et al., 2017a), as well as specific associations between dependency-related Rorschach responses and brain activity of the reward neural system (Giromini et al., 2019). The response style of the entire Rorschach protocol was related to specific neural activation. Indeed, Vitolo et al. (2021) investigated how the general complexity of given Rorschach responses (conceptualized as the psychological effort that and individual could adopt during the Rorschach task: Meyer et al., 2011) could be related to specific attentional brain networks and found significant associations between these responses and the activity of delimited brain areas concerning the dorsal attention network. More recently, Vecchio et al. (2023) summarized these and other results in a systematic review, highlighting how the involvement of the Rorschach task could be reflected by the activity of specific visual, attentional, social, and emotional brain areas.\n\nDespite the relevance of these recent studies, some problematic issues remain. First, scanner noise could compromise the ecological validity of the Rorschach test. Furthermore, the context of the scanner makes it impossible to follow standardized test procedures. Moreover, it has been suggested that neural correlates associated with different personality traits are not limited to specific brain areas but are related to large-scale dynamic interactions, including brain regions responsible for lower-level sensory functions and those responsible for higher-order cognitive functions (Adelstein et al., 2011). Given these considerations, it seems that the existing literature does not cover the aforementioned issues, focusing only on specific brain areas or administering the Rorschach during MRI scans (even if the study participants were not able to talk).\n\nTo overcome these issues, we examined the relationship between the Rorschach test and brain states in resting conditions using a particular approach. Specifically, the aims are 1) to ascertain the existence of a relationship between the CS variables and RSN, and 2) to develop a new clustering of the CS variables that reflects their association with RSNs activity.\n\n\n2. Methods\n\nIn our research design, participants underwent functional magnetic resonance imaging (fMRI) under resting conditions. In particular, they were asked to rest with their eyes open and to passively start at a point. The Rorschach test is performed outside the scanner. In this way, it was possible to obtain a more reliable measure of personality since the Rorschach test was administered in an ecological context and in full compliance with the standardization rules. Furthermore, it was possible to detect active brain patterns at rest and not during task performance.\n\nIn this study, secondary data provided anonymously were used.\n\nThe sample consisted of 24 healthy and right-handed participants who were recruited using the “snowball” sampling method. The sample was balanced by sex (12 males and 12 females) and age (mean = 29.6; sd = 6.82), with an average education level of 18 years (sd = 3.03).\n\nThe inclusion criteria were as follows: a) absence of brain accidents; b) absence of neurological and/or psychiatric pathologies; c) no use of psychotropic drugs; d) absence of pregnancy (or suspected ongoing pregnancy); e) absence of anxiety symptoms, panic attacks, and claustrophobia; f) absence of pacemakers, vascular stents, metal prostheses in the brain, heart, and large blood vessels to comply with MRI safety standards; and g) age between 20 and 40 years (to ensure comparability at the level of brain morphology).\n\nEach participant signed a self-certification as part of the informed consent, in which they stated that they had not previously received a psychiatric diagnosis and that they had never been hospitalized for this type of disorder. All procedures presented in the current study were in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.\n\nThe experimental procedure is divided into three parts. During the first phase (pre-scan phase), the following psychological tests were conducted to exclude subjects with clear psychopathological symptoms. These tests, protected by copyright, were made available by the Psychology Department of the University of Turin, for which it holds the necessary licenses.\n\n• Beck Depression Inventory - II (BDI)\n\n• State-Trait Anxiety Inventory (STAI)\n\n• SCL-90-R (Symptom Check-List - 90 Revised)\n\nIn the second part (scan phase), participants underwent resting-state fMRI scanning.\n\nFinally, the third phase (post-scan phase) took place one week after the fMRI session, which consisted of the administration of the Rorschach CS test, which is not protected by copyright.\n\nBeck Depression Inventory - II (BDI - II). The BDI-II is a self-administered questionnaire that detects the presence, severity, and intensity of depressive symptoms in individuals over 13 years of age. The Italian version was administered and validated by Ghisi et al. (2006).\n\nState-Trait Anxiety Inventory (STAI). The STAI-Y is a self-administered questionnaire that is useful for detecting the presence and intensity of anxiety symptoms in adolescents and adults. In particular, the Italian version validated by Pedrabissi and Santinello (1989) was administered.\n\nSCL-90-R (Symptom Check-List - 90 Revised). The SCL-90-R is a self-administered questionnaire that investigates a wide range of psychological problems and symptoms in individuals over 12 years of age. In particular, the Italian version validated by Sarno et al. (2011) was administered.\n\nRorschach CS. The Rorschach was administered and signed according to the Comprehensive Exner System (CS) by expert clinicians (at least five years of practice). To test the reliability of the instrument, the degree of agreement between different judges was assessed. In particular, 20 tests were randomly selected and resigned by a second independent judge. The Intraclass Correlation Coefficient indicates reliability from good (0.68 for Color-Shape responses) to excellent (1 for R). On an exploratory basis, the Rorschach variables were selected considering the trait variables most representative of the test and supported by the literature: 27 CS variables (Exner & Andronikof-Sanglade, 1992); 65 CS variables (Mihura et al., 2013). Table 1 summarizes the Rorschach variables considered in this study.\n\nNeuroimaging data analyses were performed using archival data collected for other research purposes (unpublished data). Despite procedures and data analyses referring to archival data, the findings are discussed in light of the most recent scientific literature.\n\n2.4.1 MRI data acquisition\n\nIn line with standard resting-state procedures, participants were instructed simply to keep their eyes closed, think of nothing in particular, and not to fall asleep. After each run, participants were asked if they had fallen asleep during the scan. Data from subjects with positive or doubtful answers were excluded from the study. Images were gathered using a 1.5 T INTERA™ scanner (Philips Medical Systems) with a SENSE high-field, high-resolution (MRIDC) head coil optimized for functional imaging. Two functional resting states (T2*-weighted) were acquired using echoplanar (EPI) sequences, with a repetition time (TR) of 2000 ms, an echo time (TE) of 50 ms, and a 90° flip angle. The acquisition matrix was 64x64, with a 200 mm field of view (FoV). A total of 850 volumes were acquired for the first run and a total of 450 volumes were acquired for the second run, with each volume consisting of 19 axial slices, parallel to the anterior posterior (AC–PC) commissure; slice thickness was 4.5 mm with a 0.5 mm gap. In addition to resting-state runs, 3D high-resolution T1-weighted structural images were acquired using a Fast Field Echo (FFE) sequence, with a 25ms TR, an ultrashort TE, and a 30° flip angle. The acquisition matrix was 256x256, and the FoV was 256 mm. The dataset consisted of 160 contiguous sagittal images covering the entire brain. The in-plane resolution was 1 mm × 1 mm, and the slice thickness was 1 mm (1 × 1 × 1 mm3 voxels).\n\n2.4.2 Data preprocessing\n\nAnalyses of the resting-state data were performed using the software package FSL 5.0 (Woolrich et al., 2009; Smith et al., 2004; Jenkinson et al., 2012), which is an open source comprehensive library of analysis tools for functional and structural magnetic resonance imaging, and diffusion brain imaging data ( https://fsl.fmrib.ox.ac.uk/fsl/fslwiki). The data were corrected for motion artifacts using the MCFLIRT function and spatially smoothed using a Gaussian kernel of 8-mm FWHM. High-pass temporal filtering (50s) was also performed to remove low-frequency scanner-drift artifacts. (Jenkinson et al., 2002).\n\nPreprocessed data were first co-registered to the native T1 images and then normalized into the MNI152 standard space using the FSL-FLIRT function (Jenkinson & Smith, 2001; Jenkinson et al., 2002).\n\n2.4.3 Independent component analysis (ICA)\n\nAnalyses of resting-state fMRI data were performed using independent component analysis (ICA) as implemented in the FSL MELODIC function (Beckmann & Smith, 2004). Briefly, this technique allows us to identify independent sources of signals in the brain, thereby grouping voxels showing a similar temporal profile during the resting state (i.e., the RSN). In this study, the number of independent components (ICs) to be extracted was set to 35. Seventeen of these were considered to represent noise and were discarded after visual inspection. Finally, 10 out of the 18 remaining components were retained for further analyses, as they were deemed to represent the following RSN: Default Mode Network (DMN), salience (SAL), Left Frontoparietal (LFP), Right Frontoparietal (RFP), sensorimotor (Smot), low-order visual (VIS), high-order visual (V+), cerebellar (cerebellum), temporoparietal (temporoparietal), and Supplementary Motor Area (SMA).\n\nWhen applied to multisubject data, ICA produces a unique set of independent components (ICs) for the entire sample. To obtain subject-specific ICs related to individual Rorschach responses, a dual-regression approach (Beckmann et al., 2009; Nickerson et al., 2017) was used to bring the 10 ICs (and therefore the 10 RSN) observed at the group level back to the topography of each subject.\n\nTable 2 summarizes these brain networks.\n\n2.4.4 Partial Least Square regression (PLS) analysis\n\nPatrial Least Square regression (PLS) is a machine learning technique that can be used to investigate the relationship between two sets of observations pertaining to the same group of subjects but based on different variables. This is achieved by means of a dimensionality reduction technique that allows to identify the so called “latent factors” that can explain both two sets of variables used (Chen et al., 2019). In more technical terms, the goal of the Partial Least Square Regression (PLS) is to predict Y from X and to describe their common structure. In this specific case,X is a matrix in which each row represents a different participant and each column represents the activity in a given voxel of the brain. Y is again a subject × variable matrix, with the variables being CS Rorschach scores.\n\nTherefore, the aim of PLS in our study is to predict Rorschach variables on the basis of resting-state activity and to describe their common structure. Besides producing the latent factors, the output also returns the so called “factors loading.” These represent the strength of the association between the computed latent factors and the variables originally measured for the sample and used as inputs for the analysis.\n\nThe first calculation step is the mean centering of the data X then the relationship between the columns of X and Yis obtained by a cross-product as:\n\nThe SVD decompose R into three matrices:\n\nThe latent variables of X are given by:\n\nThis matrix shows how the brain activity relates to each observation.\n\nThen, the loading of X (the projection of X onto the space of T) is given by\n\nP describe the voxels as explained by the latent variables.\n\nIn the same manner for the behavioral variables stored in Y we have:\n\nStatistical inference consists of two separate analyses. Fixed effects and random effects models. For the fixed effects model, the quality of the regression of the L latent variables is obtained by computing the predicted matrix Ŷ and then measuring the similarity with the original data Y. The most popular measure of similarity is the residual sum of squares, defined as:\n\nTo generalize the results to the population of interest, the random effects model, it is necessary to estimate the generalization capacity of the PLSR model. In this case, a standard parametric approach can be used; therefore, the model was evaluated using the jackknife method.\n\nIn the jackknife method, each observation is excluded from the data, and the remaining set of data constitutes the learning set. Each of these sets is used to perform a PLSR that is used to predict the left-out observation. The predicted observations were stored in a matrix called Y˜. The quality of the model was evaluated as the similarity between Y˜ and Y. In this case, the best measure is the residual sum of squares defined as\n\nFor a random model, it is critical to determine the optimal number of latent variables for analysis. A good approach (Tenenhaus, 1998) starts by adding a latent variable, one at a time, and computing the ratio for the l-th latent variable defined as\n\nWhen the number of correct latent variables was obtained, the confidence intervals of the predicted values were derived using a bootstrap statistic. In a bootstrap statistic, a large sample is obtained by drawing observations with replacements from the set of original data. The PLS distribution obtained using these observations was used to estimate the sampling distribution of the parameters.\n\n2.4.3 Cluster analysis\n\nAs explained before, PLS allowed us to understand the relationship between resting state activity in 10 RSN and 27 CS scoring variables of the Rorschach signature. However, resting-state information could also be useful to better elucidate the relationship between CSs. Clustering methods can be used to group items that show a high degree of similarity with a given dimension (Cauda et al., 2010; Manuello et al., 2022). In this specific case, the similarity among the CSs was based on the variance explained by them for the activity profile of the RSNs. In practice, a vector called “network profile” was created for each CS. Each value of this vector represents the variance explained by the specific CS for each of the ten RSN. The obtained network profiles were then organized into a CSs × RNS matrix (i.e., 27 rows for 10 columns) that was used as the input for the hierarchical clustering analysis. The between-row distance (i.e., between CSs) was computed using Euclidean distance as a metric, whereas clusters were created using Ward’s linkage (Ward, 1963).\n\n\n3. Results\n\nPLS regression showed that by means of three latent factors, the 27 considered Rorschach variables could explain the activity observed in ten ICs identified through ICA, which had been retained for further analyses as they were deemed to represent some canonical RSNs.\n\nLatent factor 1 was found to explain the variance in each of the considered RSN, with a peak at approximately 40% for the SMOT network. Latent factor 2 explained a much reduced amount of variance for the SAL, LFP, and CEREB networks. This was also the only network that showed some contribution from latent factor 3 (Figure 1).\n\nNote: Values on the y-axis represent the percentage of explained variance; VIS = low-order visual; DMN = default mode network; SMOT = sensorimotor; LFP = left frontoparietal; SAL = salience; RFP = right frontoparietal; SMA = supplementary motor area; TEMP-PAR = temporo-parietal (auditory); V+ = high-order visual; CEREB = cerebellar.\n\nIn addition to estimating the variance explained by each of the three latent factors, PLS regression allowed the quantification of the relationship between each variable and the obtained latent factors. It was therefore possible to observe how the 27 CSs were related to each of the three latent factors, and at the same time, which voxels across the brain each factor is relevant for (Figures 2 to 11). Notably, for all ten RSNs considered, latent factor 1 showed a loading pattern that closely resembled the original brain network. This means that the relationship between a given RSN and the 27 CSs captured by the latent factor involved the whole RSN in a way comparable to how it had been observed during the resting state and not only by the contribution of a few sparse voxels across the brain. Moreover, even when latent factors 2 and 3 were involved, the significant voxels were still localized in a coherent manner with the expected spatial pattern for canonical RSNs.\n\nRegarding the loadings on CSs, the latent factors showed that most of the considered RSNs were associated with only a few of them.\n\nIn fact, the DMN showed the contribution of latent factor 1 only, with a unique loading on the Rorschach variable HVI_cont (Figure 2). Similarly, the TEMP-PAR (auditory) network showed the contribution of latent factor 1 only, with a unique loading on the Rorschach variable WSumC (Figure 3).\n\nBoth visual RSNs (i.e., VIS and V+) show the contribution of latent factor 1 only, with a unique loading on Rorschach variables DEPI_cont for the former (Figure 4) and loadings on CDI_cont and GHR for the latter (Figure 5).\n\nThe networks involved in sensory-motor processing (i.e., SMOT and SMA) again show the contribution of latent factor 1 only, with loadings on Rorschach variables PTI_cont and M- for the former (Figure 6) and HVI_cont and M for the latter (Figure 7).\n\nFinally, RFP shows the contribution of latent factor 1 only, with loading on the Rorschach variables PTI_cont, M-, and WSum6 (Figure 8). Contrary to its right counterpart, the LFP showed the contributions of both latent factors 1 and 2. The loadings for factor 1 were on Rorschach variables R and CritCont, whereas HVI_Cont, M, 2, and GHR were associated with latent factor 2 (Figure 9).\n\nIn addition, the SAL network showed the contribution of both latent factors 1 and 2: the only loading for the former was on the Roarshach variable Art, while the loadings for Factor 2 were on the Rorschach variables An, CDI, CritCont, GHR, and M. (Figure 10).\n\nThe cerebellar network is the only network that shows the contribution of all three latent factors. The loading on Rorschach variables was onCDI_cont for latent factor 1; AdjD_cont, R, F, AB, and GHR for latent factor 2; and HVI_cont, M, 2, Wsum6, and PHR for latent factor 3 (Figure 11).\n\nThe dendrogram obtained through cluster analysis clearly suggested the existence of four clusters across the 27 CSs (Figure 12). The widest cluster contained 12 CSs, whereas PTI_cont and M- comprised the smallest cluster.\n\nNote: For Rorschach variables abbreviations, please see Table 1.\n\n\n4. Discussion\n\nThe Rorschach test is one of the most popular assessment tools used in clinical settings for psychopathology and personality assessments. However, it is not yet clear how the verbal replies offered by patients, and captured through signatures, relate to neural activity. Previous investigations have attempted to use task-fMRI to solve this issue. However, as mentioned above, this approach has technical criticalities that could bias the results. For this reason, the present study adopted the resting state instead to be associated with scoring of the Rorschach test administered at a later time. Notably, resting-state networks are thought to represent a more stable modality of each individual’s brain activity, as this is not affected or guided by the task implemented during the MRI scan.\n\nFrom a neurobiological standpoint, our results underlined specific brain activity patterns associated with the given Rorschach variables. First, we observed that DMN activity was significantly negatively correlated with HVI. This variable reflects relational and cognitive style, thus representing psychological processes such as control, emotional and relational distance, and alert and distrust towards the environment (Exner, 1991; Exner & Erdberg, 2005; Mihura et al., 2013). In other words, it could represent a sort of hyper-vigilance process towards the inner and external worlds. It is noteworthy that the HVI variable showed a positive correlation with the SAL network activity, which came along with variables that represent a kind of social attention pointed towards representations of self and interpersonal relationships (i.e., An, CritCont, GHR, and M, grouped in the “Factor 2”).\n\nThe DMN is involved in the monitoring of somato-emotional and vegetative information, feelings and body posture, free association of thoughts, inner speech, mental images, emotions, planning of future events, interpretation and prediction of environmental requirements, autobiographical memories, working memory, theory of mind, integration, creativity, self-referential thought, and integration between self and environment (Cozolino, 2020). In contrast, the SAL Network is involved in the interoception of feelings associated with gratification and consists of synchronous activation of the insula, anterior cingulate cortex, and ventral striatum (Pace-Schott & Picchioni, 2017). Moreover, it is involved in understanding others’ intentions (Rijpma et al., 2021) and in self-recognition from first- and third-person perspectives, a critical process for the development of self-consciousness (Asakage & Nakano, 2023). According to the Rorschach literature, it seems more appropriate to consider the HVI construct through the DMN-SAL activation axis. Indeed, the mutually excluding activation of the DMN and SAL (Goulden et al., 2014; Sridharan et al. 2008), well describes the circularity of psychic investment of the hyper-vigilant person. Hypervigilance may be represented by two components: the first one is more internally directed and is likely supported by a series of functions in which the DMN is involved; the second one is more other-directed and may be supported by SAL activity (Koch et al., 2016).\n\nHypervigilant subjects, because of their need to maintain interpersonal control, invest a high amount of energy to ensure that all elements in the field are under their control. The result is that this self-directive cognitive activity, supported by the DMN, is chaotic and inefficient because they are stuck in their flood of thoughts (rumination). DMN activity is associated with high rumination in patients (Boyd et al., 2018). It is likely that depressed subjects have high HVI scores because of their tendency to ruminate in a rigid and persistent way about a world that is perceived as threatening and hostile. In other words, it is likely that hypervigilant subjects respond to their sense of fragility by overcontrolling everything. At the same time, hypervigilant people pay attention to external stimuli, which have to remain under their control (Richards et al., 2014).\n\nOur analysis underlined the involvement of the TEMP-PAR network. Although it has historically been associated with language processes (Rosazza & Minati, 2011), it has recently been associated with emotion regulation processes (Buhle et al., 2014; Kohn et al., 2014; Morawetz et al., 2017; Vitolo et al., 2022). Indeed, it has been outlined how emotion regulation processes, despite their strong association with prefrontal activity (Etkin et al., 2015; Goldin et al., 2008), could recruit large portions of parietal and temporal regions (Morawetz et al., 2017; Ochsner et al., 2012; Sripada et al., 2014), because of the additional attentional and re-interpretative processes involved in modulation processes (Morris et al., 2014; Ochsner et al., 2012). Interestingly, the activity of the TEMP-PAR network was found to be related, in our analyses, to the presence of the Rorschach variable WSumC, which represent the weighted sum of the responses in which the respondents “use” the inkblots’ colors to provide a rationale for their responses (Exner & Erdberg, 2005). During the Rorschach task, using colorful details to provide responses could indicate the way in which the respondents express their emotions, thus performing a sort of self-modulation of themselves (Exner, 2003; Malone et al., 2013). Pushing forward to these concepts, the Rorschach variable WSumC represents the presence of emotional modulation processes (Exner, 2003). Thus, its association with a brain network involved in emotion regulation processes, such as the TEMP-PAR network, as outlined in our analyses, could foster these interpretations.\n\nMoving forward, our analyses revealed an association between certain Rorschach variables and the activity of the visual network in both its lower-order (VIS) and higher-order (V+) components. Although this network is particularly active during visual tasks (Rosazza & Minati, 2011), it has been observed that activation of the visual network could modulate BOLD fluctuations in resting-state activity (Stevens et al., 2010). Our analyses outlined how the resting-state activity of both visual components was associated with the Rorschach variables DEPI, CDI, and GHR. Interestingly, DEPI and CDI were positively associated with both visual components, whereas GHR was negatively associated with the high-order visual component (V+). According to the Rorschach literature, the CDI variable could be present in individuals who might experience a sort of immaturity and frustration in living social situations, strongly centered on themselves with a high need for the other, thus being less able to experience a relationship in a rewarding manner (Exner, 2003; Exner & Erdberg, 2005). In contrast, the GHR variable represents the exact opposite, indicating individuals that might have internalized good representations of social relationships (Exner, 2003; Exner & Erdberg, 2005). Moreover, the DEPI variable was elaborated with the intention of detecting typical depressive functioning (Exner, 2003; Exner & Erdberg, 2005), and along with CDI, it was found to be associated with depressive symptoms (Carlson et al., 1997), thus representing a marker for depressive tendencies (Mihura et al., 2013). Notably, patients with depression have been found to show patterns of decreased functional connectivity in the visual areas that comprise the visual network (Lu et al., 2020). Given that high values of DEPI and CDI and low values of GHR could be associated with depressive tendencies (Mihura et al., 2013), the observed association between resting activation of the visual components and the aforementioned Rorschach variables (i.e., DEPI, CDI, and GHR) could be interpreted in light of the abnormal functional activity patterns of the visual areas observed in depressive patients (Lu et al., 2020).\n\nOther relevant findings concern the SMOT network, the activation of which has been historically associated with covert and overt movement, namely motor imagery and passive movement (Jeannerod, 1994; Naito et al., 2002; Stippich et al., 2002). Interestingly, the resting activity if this network was negatively associated, in our analysis, with the presence of Rorschach variables M- and PTI that, as mentioned above, is linked to abnormal manifestations of thoughts and perceptions distortions (Exner, 1991; Exner & Erdberg, 2005; Mihura et al., 2013). More intriguingly, reduced intrinsic activity of SMOT network was found in patients with schizophrenia (Kaufmann et al., 2015). Our results are in line with these considerations, outlining how a more functionally connected SMOT network is associated with a less severe marker of thought disorders. Moreover, motor areas encompassed in the SMA network showed a significant negative association with the HVI Rorschach variable, which serves as an indicator of hypervigilance (Exner, 1991; Exner & Erdberg, 2005; Mihura et al., 2013). Interestingly, Breckel et al. (2011) reported that decreased activity of motor areas (e.g., the SMA) was associated with prolonged exposure to a vigilance task, suggesting that this decreased activity could be interpreted as a function of the time interaction on vigilance processes. Again, our results concerning the negative association between HVI and SMA activity seem to be in line with the aforementioned findings.\n\nOur analyses revealed involvement of the Frontoparietal Network (FPN) in both the right (RFP) and left (LFP) components. Historically, FPN activity has been associated with different functions such as memory (Damoiseaux et al., 2006), attention (Smith et al., 2009), language (Dosenbach et al., 2007; Fox et al., 2005), and visual processes (De Luca et al., 2006). It is noteworthy that FPN is associated with mentalization and reflective processes of the self and others (Lieberman, 2007; Luyten et al., 2020). Given these findings, our results relying on associations between LFP and Rorschach variables that outlined the presence of attentive and mental processes of representations of self and interpersonal relationships (i.e., HVI, M, 2, and GHR, grouped in the “Factor 2”) were not surprising. In contrast, abnormal functional intrinsic and extrinsic connectivity has been found in the right components of FPN in psychotic patients (Baker et al., 2014), as well as in individuals at high risk for mental illness (Peeters et al., 2015; Schmidt et al., 2014). Our analyses revealed that the right components of FPN were negatively associated with Rorschach variables M-, PTI, and WSum6, which could be considered an indicator of thoughts and perception disorganization, thus representing a marker for the presence of a possible psychotic disorder (Exner, 1991; Exner & Erdberg, 2005; Mihura et al., 2013).\n\nThe hypervigilant other-directed component can refer to all assets managed by the SAL, including attention to external stimuli.\n\nFactor 1 (Art) explains most of the variance (38%) of the salience network consistently with the processes of attention to detail and integration of visual-spatial perceptual elements linked to visual-motor coordination in order to integrate salient stimuli. In this sense, we can ask ourselves about the construct of the intellectualization index (2AB + Art + Ay), which indicates the process through which the subject tries to reduce or neutralize the intensity of emotions, focusing on the formal features of the percept.\n\nFactor 2 (An, CDI, CritCont, GHR, M) explains approximately 10% of the variance in the salience network. CDI indicates difficulty in the area of control and sociality; it characterizes socially immature subjects who have problems interacting with the external environment in emotionally complex social situations. The An variable indicates concerns related to body image (Mihura et al., 2013). The CritCont variable identifies depressive content, needs, and urgencies that are typically inhibited, minimized, or expressed indirectly through an adaptive way of thinking. GHR is an effective approach for interpersonal relationships. Variable M captures the subject’s ability to empathically identify with others. Overall, the variables that explain the large state brain network of SAL can be referred to as social attention processes linked to self-image (CDI, An, and CritCont) and interpersonal relationships (GHR). These data are coherent with spatial detail attention (art) and top-down processes of primary sensory inputs that are typical of this network. In fact, the Salience System is active during the detection of signals, attention to spatial details, and hand-eye coordination. It is also associated with top-down control processes of primary sensory inputs (Friston & Büchel, 2000; Lachaux et al., 2005).\n\nOne of the most valuable results concerns the cerebellar network, the activity of which was found to be associated with several Rorschach variables grouped into three main latent factors. Factor 1 was represented by the CDI variable alone, which, as mentioned before, could be a marker for depressive tendencies (Mihura et al., 2013). Factor 2 comprises five Rorschach variables, namely AdjD, R, F, AB, and GHR, which, grouped together, could represent a sort of tendency towards reality simplification (Exner & Erdberg, 2005). Finally, Factor 3 entails the other five Rorschach variables: HVI, M, 2, WSum6, and PHR. When grouped together, these variables could represent the behaviors of hyper-vigilant individuals, particularly those who are warried about their relationships and the emotional contexts in which they are usually embedded. In these contexts, individuals are typically involved in emotional processing and modulation, thus paying attention to other people and the external environment in general (Exner, 2003; Exner & Erdberg, 2005). Regarding the cerebellar network, it has recently been demonstrated that the cerebellar regions associated with motor functions are distinct from those serving non-motor and cognitive functions (Stoodley & Schmahmann, 2009). Historically associated with coordination processes of motor behaviors (Bastian et al., 1999), the cerebellum has recently been shown to play a role in emotional processes (Adamaszek et al., 2017; Baumann & Mattingley, 2012; Schutter & van Honk, 2005), as well as regulation of emotions (Schutter & van Honk, 2009; Turner et al., 2007). Resting-state functional connectivity studies have shown several associations between cerebellar activity and brain regions involved in emotion regulation processes, such as the salience network brain regions (Seeley et al., 2007), amygdala (Roy et al., 2009; Sang et al., 2012), and insular and cingulate cortices (Allen et al., 2005). These findings could explain the association between cerebellar activity and Factor 2 (positive association) and Factor 3 (negative association). Indeed, given that higher cerebellar activity could be associated with both cognitive and emotional processes (Stoodley et al., 2012; Stoodley & Schmahmann, 2018), associations with factors 2 and 3 could explain these considerations. In contrast, Factor 1 (i.e., the Rorschach variable CDI) showed a positive association with cerebellar activity. Given that CDI could represent a marker of depressive tendencies (Mihura et al., 2013), the positive association with cerebellar activity could assume a counterintuitive meaning, in the extent of emotional and cognitive impairments are often associated to depressivepsychotic manifestations (Drevets, 2001). However, it has been recently found that patients with depression may show increased patterns of intrinsic functional connectivity in the cerebellum (Dai et al., 2023). Although these findings could explain the positive association between CDI and cerebellar activity, it seems to be in contrast with the aforementioned findings. Given these contradictory results, our interpretations should be interpreted with caution, probably considering them only at the speculative level, waiting for further investigations.\n\nThe aforementioned results pointed out interesting considerations, first concerning a clinical perspective that could embrace Rorschach’s variable interpretations. Rorschach variables that shared a similar network profile were grouped using cluster analysis. The four clusters of variables have interesting diagnostic meanings. In particular, the first one (composed of PTI and M-) could be central from both psychometric and diagnostic points of view, in that it detects the effects of cognitive processing and possible distortions that may affect the clarity of thoughts. The second (formed by DEPI, SCON, DScore, Xy, Ay, r, FQ-, WSumC, Art, 2, WSum6, and PHR) might refer to depressive features, emotion regulation processes, and representations of self and others. The third one (which comprises AdjD, F, AB, R, and GHR) is more heterogeneous and may be associated with a set of features arranged on a continuum from performance and perception components vs symbolic thought. Finally, the fourth and final clusters (composed of HVI, M, CDI, An, and CritCont) can be traced back to characteristics related to social anxiety, worry, and hypervigilance.\n\nTaken together, these findings provide additional evidence indicating that, in some specific profiles, interesting points of convergence emerge between the Rorschach nomological network and RSN. Within this view, our study can be grouped with other studies that attempt to provide evidence-based findings regarding the validity and reliability of the Rorschach test (e.g., e.g., Giromini, Viglione, Pineda et al., 2017a; Giromini, Viglione, Zennaro et al., 2017b; Giromini et al., 2019; Ishibashi et al., 2016; Vitolo et al., 2021). This could help to shed new light on both the Rorschach test, especially the dialogue between neuroscience and clinical practice, and again between mind and brain. Data from this research could provide a starting point for further exploration of the hypotheses that they have generated.\n\nFinally, this study has some limitations. First, the analyzed sample consisted of only 24 participants. However, while it is generally true that neuroimaging has been shifting in recent years towards a big-data approach, our sample is in line with the median size currently used in the field (Szucs & Ioannidis, 2020). Indeed, similar empirical studies on the neural correlates of Rorschach responses included less than 30 subjects (Giromini et al., 2017a, 2017b, 2019; Vitolo et al., 2021). The second element of concern may be related to the RSN identification. ICA is the most commonly used technique to extract patterns of brain activity at rest, but it does not provide standardized labels for the observed components. Therefore, we followed the general and accepted procedure of manual identification carried out by researchers (F. C. and T.C.) with highly specific experience for resting-state data. Moreover, dual regression allowed for a correct return from the RSNs observed at the group level to the subject-specific spatial pattern.\n\n\n5. Conclusions\n\nOur data show a strong relationship between Neuroimaging and Rorschach. These results support the possibility of investigating the psychic functions underlying Rorschach variables by studying the activation of different brain areas under resting conditions. Furthermore, this type of research would make it possible to extend the use of neuroimaging techniques to include further psychological testing. Neuroscience and clinical psychology share the same object of study: the human mind and its manifestations.\n\nThe fracture between clinical practice and neuroscience was partly caused by Freud’s lack of attempt to corroborate his theory with the aid of neuroscience, mainly because of the absence of adequate tools (Northoff, 2012). However, today’s neuroimaging techniques have completely revolutionized the way of conceiving the study of the brain, allowing the movement of an anatomical-structural vision of brain mechanisms to a more functional one based on the connecting networks between the various areas of the brain. As pointed out by Northoff (2012), Freud would probably be very interested in neuroscience today, finally having tools to investigate the psyche in a fairly sophisticated way, and therefore, similar to how he imagined it.\n\nMoreover, neuroimaging methods can be employed to investigate psychopathological risk factors in both typical and atypical brain maturation processes (Díaz-Arteche & Rakesh, 2020). Thus, our study could be included in this growing integrative perspective, promoted even in other studies (e.g., Giromini et al., 2017a, 2017b, 2019; Ishibashi et al., 2016; Vitolo et al., 2021), and is useful in clinical practice. Indeed, detecting information about problem-solving and perceptual processes usually activated during the Rorschach task could help future researchers and clinicians better understand the interplay between neurobiological and psychological processes.\n\n\nEthics statement\n\nThe study was approved by the Ethics Committee of the University of Valle d’Aosta (protocol no. 97/2024 dated January 8, 2024). At the time of data collection (2011), the Ethics Committee had not yet been established. It was established by Decree of the President of the University Council No. 2, Protocol No. 7475/II26, on August 28, 2015. However, the research complied with the Codice Etico AIP (Ethical Code for Research in Psychology, Italian Psychological Association) and the provisions of Italian privacy and data protection laws (L. 196/2003). All procedures performed in the present study were thus in accordance with the ethical standards of the institutional and/or national research commission and the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards. In addition, we obtained a favourable opinion from the Ethics Committee of the University of Valle d’Aosta (protocol no. 97/2024 of January 8, 2024) for the analysis of the archival data, and given the observational nature of the study, the same Ethics Committee considered the collection of these data to be compatible with ethical research standards.\n\n\nConsent statement\n\nThe research utilized archive data provided by the Tiarè Association, Mental Health Services. From the data available to us, it is evident that informed consent was requested in written form.\n\n\nAuthor contributions\n\nStefania Cristofanelli:\n\nRoles: Conceptualization, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing\n\nEnrico Vitolo:\n\nRoles: Data Curation, Validation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing\n\nAlessandro Zennaro:\n\nRoles: Conceptualization, Supervision, Resources, Writing – Original Draft Preparation\n\nFranco Cauda:\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Visualization, Writing – original draft preparation\n\nTommaso Brischetto Costa:\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Visualization, Writing – original draft preparation\n\nEleonora Centonze:\n\nRoles: Writing – Original Draft Preparation, Writing – Review & Editing\n\nGiorgia Baccini:\n\nRoles: Writing – Original Draft Preparation, Writing – Review & Editing\n\nJordi Manuello:\n\nRoles: Validation, Writing – Review & Editing\n\nLaura Ferro:\n\nRoles: Conceptualization, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing\n\nAll authors reviewed the results and approved the final version of the manuscript.", "appendix": "Data availability statement\n\nThe data that support the findings of this study are available from the TIARÉ Association for Mental Health. Restrictions apply to the availability of these data, which were used under license for this study. TIARÉ Association for Mental Health collected these data for clinical and research purposes and has thus formalized agreement and authorization on the following points: “Types of data identified and processed by the research group; Operations and procedures performed during data collection and processing; Scientific dissemination of the emerged data; Relevance of the project as being of public/social interest in addition to scientific interest”. The Ethics Committee has therefore expressed a favorable opinion on the appropriateness of the specific measures identified to protect the fundamental rights and interests of the involved parties. Data are available upon reasonable request from the corresponding authors (Enrico Vitolo, Stefania Cristofanelli) luca.ortolan@associazionetiare.org with the permission of the TIARÉ Association for Mental Health.\n\n\nReferences\n\nAdamaszek M, D’Agata F, Ferrucci R, et al.: Consensus Paper: Cerebellum and Emotion. Cerebellum (London, England). 2017; 16(2): 552–576. PubMed Abstract \| Publisher Full Text\n\nAdelstein JS, Shehzad Z, Mennes M, et al.: Personality is reflected in the brain’s intrinsic functional architecture. PLoS One. 2011; 6(11): e27633. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllan PG, Briggs RG, Conner AK, et al.: Parcellation-based tractographic modeling of the dorsal attention network. Brain Behav. 2019; 9(10): e01365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen G, McColl R, Barnard H, et al.: Magnetic resonance imaging of cerebellar-prefrontal and cerebellar-parietal functional connectivity. NeuroImage. 2005; 28(1): 39–48. PubMed Abstract \| Publisher Full Text\n\nAsakage S, Nakano T: The salience network is activated during self-recognition from both first-person and third-person perspectives. Hum. Brain Mapp. 2023; 44(2): 559–570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker JT, Holmes AJ, Masters GA, et al.: Disruption of cortical association networks in schizophrenia and psychotic bipolar disorder. JAMA Psychiatry. 2014; 71(2): 109–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBastian AJ, Mugnaini E, Thach WT: Cerebellum.Zigmond MJ, Bloom FE, Landis SC, et al., editors. Fundamental neuroscience. San Diego (CA): Academic Press; 1999; pp. 973–992.\n\nBaumann O, Mattingley JB: Functional topography of primary emotion processing in the human cerebellum. NeuroImage. 2012; 61(4): 805–811. PubMed Abstract \| Publisher Full Text\n\nBeckmann CF, Mackay CE, Filippini N, et al.: Group comparison of resting-state FMRI data using multi-subject ICA and dual regression. NeuroImage. 2009; 47(Suppl 1): S148. Publisher Full Text\n\nBeckmann CF, Smith SM: Probabilistic independent component analysis for functional magnetic resonance imaging. IEEE Trans. Med. Imaging. 2004; 23(2): 137–152. Publisher Full Text\n\nBiswal BB, Mennes M, Zuo XN, et al.: Toward discovery science of human brain function. Proc. Natl. Acad. Sci. USA. 2010; 107(10): 4734–4739. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiswal B, Yetkin FZ, Haughton VM, et al.: Functional connectivity in the motor cortex of resting human brain using echo-planar MRI. Magn. Reson. Med. 1995; 34(4): 537–541. PubMed Abstract \| Publisher Full Text\n\nBoyd JE, Lanius RA, McKinnon MC: Mindfulness-based treatments for posttraumatic stress disorder: a review of the treatment literature and neurobiological evidence. J. Psychiatry Neurosci. 2018; 43(1): 7–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreckel TP, Giessing C, Thiel CM: Impact of brain networks involved in vigilance on processing irrelevant visual motion. NeuroImage. 2011; 55(4): 1754–1762. PubMed Abstract \| Publisher Full Text\n\nBuckner RL, Vincent JL: Unrest at rest: default activity and spontaneous network correlations. NeuroImage. 2007; 37(4): 1091–1096. PubMed Abstract \| Publisher Full Text\n\nBuhle JT, Silvers JA, Wager TD, et al.: Cognitive reappraisal of emotion: a meta-analysis of human neuroimaging studies. Cereb. Cortex. 2014; 24(11): 2981–2990. Publisher Full Text\n\nCanli T, Amin Z: Neuroimaging of emotion and personality: scientific evidence and ethical considerations. Brain Cogn. 2002; 50(3): 414–431. PubMed Abstract \| Publisher Full Text\n\nCarlson CF, Kula ML, St Laurent CM: Rorschach revised DEPI and CDI with inpatient Major Depressives and Borderline Personality Disorder with major depression: validity issues. J. Clin. Psychol. 1997; 53(1): 51–58. PubMed Abstract \| Publisher Full Text\n\nCauda F, Geminiani G, D’Agata F, et al.: Functional connectivity of the posteromedial cortex. PLoS One. 2010; 5(9): e13107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen K, Azeez A, Chen DY, et al.: Resting-State Functional Connectivity: Signal Origins and Analytic Methods. Neuroimaging Clin. N. Am. 2020; 30(1): 15–23. PubMed Abstract \| Publisher Full Text\n\nChen C, Cao X, Tian L: Partial least squares regression performs well in MRI-based individualized estimations. Front. Neurosci. 2019; 13: 1282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCozolino L: The Pocket Guide To Neuroscience For Clinicians. W.W. Norton & Company, Inc.; 2020.\n\nDai P, Zhou X, Xiong T, et al.: Altered Effective Connectivity Among the Cerebellum and Cerebrum in Patients with Major Depressive Disorder Using Multisite Resting-State fMRI. Cerebellum (London, England). 2023; 22(5): 781–789. PubMed Abstract \| Publisher Full Text\n\nDamoiseaux JS, Rombouts SA, Barkhof F, et al.: Consistent resting-state networks across healthy subjects. Proc. Natl. Acad. Sci. 2006; 103(37): 13848–13853. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Luca M, Beckmann CF, De Stefano N, et al.: fMRI resting state networks define distinct modes of long-distance interactions in the human brain. NeuroImage. 2006; 29(4): 1359–1367. PubMed Abstract \| Publisher Full Text\n\nDeYoung CG, Hirsh JB, Shane MS, et al.: Testing predictions from personality neuroscience. Brain structure and the big five. Psychol. Sci. 2010; 21(6): 820–828. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDíaz-Arteche C, Rakesh D: Using neuroimaging to predict brain age: insights into typical and atypical development and risk for psychopathology. J. Neurophysiol. 2020; 124(2): 400–403. PubMed Abstract \| Publisher Full Text\n\nDosenbach NU, Fair DA, Miezin FM, et al.: Distinct brain networks for adaptive and stable task control in humans. Proc. Natl. Acad. Sci. USA. 2007; 104(26): 11073–11078. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrevets WC: Neuroimaging and neuropathological studies of depression: implications for the cognitive-emotional features of mood disorders. Curr. Opin. Neurobiol. 2001; 11(2): 240–249. PubMed Abstract \| Publisher Full Text\n\nDronkers NF, Wilkins DP, Van Valin RD, et al.: Lesion analysis of the brain areas involved in language comprehension. Cognition. 2004; 92: 145–177. PubMed Abstract \| Publisher Full Text\n\nEtkin A, Büchel C, Gross JJ: The neural bases of emotion regulation. Nat. Rev. Neurosci. 2015; 16(11): 693–700. Publisher Full Text\n\nExner JE: The Rorschach: A comprehensive system—Vol. 2. Interpretation. 2nd ed.New York, NY: Wiley; 1991.\n\nExner JE: The Rorschach: A comprehensive system. Volume 1: Basic foundations and principles for interpretation. 4th ed.New York, NY: John Wiley; 2003.\n\nExner JE Jr, Andronikof-Sanglade A: Rorschach changes following brief and short-term therapy. J. Pers. Assess. 1992; 59(1): 59–71. PubMed Abstract \| Publisher Full Text\n\nExner JE, Erdberg P: The Rorschach: A comprehensive system. Volume 2: Advanced interpretation. Hoboken, NJ: Wiley; 2005.\n\nFox MD, Snyder AZ, Vincent JL, et al.: The human brain is intrinsically organized into dynamic, anticorrelated functional networks. Proc. Natl. Acad. Sci. USA. 2005; 102(27): 9673–9678. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriston KJ, Büchel C: Attentional modulation of effective connectivity from V2 to V5/MT in humans. Proc. Natl. Acad. Sci. USA. 2000; 97(13): 7591–7596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhisi M, Flebus GB, Montano A, et al.: BDI-II. Beck Depression Invetory – II. Manuale. Seconda edizione. Firenze, ITA: Giunti Psychometrics; 2006.\n\nGiromini L, Viglione DJ, Pineda JA, et al.: Human movement responses to the rorschach and mirroring activity: an fMRI study. Assessment. 2017a; 26(1): 56–69. PubMed Abstract \| Publisher Full Text\n\nGiromini L, Viglione DJ, Vitolo E, et al.: Introducing the concept of neurobiological foundation of Rorschach responses using the example of Oral Dependent Language. Scand. J. Psychol. 2019; 60(6): 528–538. PubMed Abstract \| Publisher Full Text\n\nGiromini L, Viglione DJ, Zennaro A, et al.: Neural activity during production of rorschach responses: an fMRI study. Psychiatry Res. Neuroimaging. 2017b; 262: 25–31. PubMed Abstract \| Publisher Full Text\n\nGoldin PR, McRae K, Ramel W, et al.: The neural bases of emotion regulation: reappraisal and suppression of negative emotion. Biol. Psychiatry. 2008; 63(6): 577–586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoulden N, Khusnulina A, Davis NJ, et al.: The salience network is responsible for switching between the default mode network and the central executive network: replication from DCM. NeuroImage. 2014; 99: 180–190. PubMed Abstract \| Publisher Full Text\n\nGuell X, Gabrieli JDE, Schmahmann JD: Triple representation of language, working memory, social and emotion processing in the cerebellum: convergent evidence from task and seed-based resting-state fMRI analyses in a single large cohort. NeuroImage. 2018; 172: 437–449. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGusnard DA, Akbudak E, Shulman GL, et al.: Medial prefrontal cortex and self-referential mental activity: relation to a default mode of brain function. Proc. Natl. Acad. Sci. USA. 2001; 98(7): 4259–4264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIshibashi M, Uchiumi C, Jung M, et al.: Differences in brain hemodynamics in response to achromatic and chromatic cards of the rorschach: a fMRI study. Rorschachiana. 2016; 37(1): 41–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeannerod M: The representing brain: Neural correlates of motor intention and imagery. Behav. Brain Sci. 1994; 17(2): 187–202. Publisher Full Text\n\nJenkinson M, Bannister P, Brady M, et al.: Improved optimization for the robust and accurate linear registration and motion correction of brain images. NeuroImage. 2002; 17(2): 825–841. PubMed Abstract \| Publisher Full Text\n\nJenkinson M, Beckmann CF, Behrens TE, et al.: Fsl. NeuroImage. 2012; 62(2): 782–790. Publisher Full Text\n\nJenkinson M, Smith S: A global optimisation method for robust affine registration of brain images. Med. Image Anal. 2001; 5(2): 143–156. PubMed Abstract \| Publisher Full Text\n\nKaufmann T, Skåtun KC, Alnæs D, et al.: Disintegration of Sensorimotor Brain Networks in Schizophrenia. Schizophr. Bull. 2015; 41(6): 1326–1335. Publisher Full Text\n\nKoch SB, van Zuiden M , Nawijn L, et al.: Aberrant resting-state brain activity in posttraumatic stress disorder: A meta-analysis and systematic review. Depress. Anxiety. 2016; 33(7): 592–605. PubMed Abstract \| Publisher Full Text\n\nKohn N, Eickhoff SB, Scheller M, et al.: Neural network of cognitive emotion regulation--an ALE meta-analysis and MACM analysis. NeuroImage. 2014; 87: 345–355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoyama MS, Kelly C, Shehzad Z, et al.: Reading networks at rest. Cerebral Cortex (New York, N.Y.: 1991). 2010; 20(11): 2549–2559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLachaux JP, George N, Tallon-Baudry C, et al.: The many faces of the gamma band response to complex visual stimuli. NeuroImage. 2005; 25(2): 491–501. PubMed Abstract \| Publisher Full Text\n\nLanssens A, Pizzamiglio G, Mantini D, et al.: Role of the dorsal attention network in distracter suppression based on features. Cogn. Neurosci. 2020; 11(1-2): 37–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLieberman MD: Social cognitive neuroscience: a review of core processes. Annu. Rev. Psychol. 2007; 58: 259–289. Publisher Full Text\n\nLu F, Cui Q, Huang X, et al.: Anomalous intrinsic connectivity within and between visual and auditory networks in major depressive disorder. Prog. Neuro-Psychopharmacol. Biol. Psychiatry. 2020; 100: 109889. PubMed Abstract \| Publisher Full Text\n\nLund MJ, Alnæs D, Rokicki J, et al.: Functional connectivity directionality between large-scale resting-state networks across typical and non-typical trajectories in children and adolescence. PLoS One. 2022; 17(12): e0276221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuyten P, Campbell C, Allison E, et al.: The Mentalizing Approach to Psychopathology: State of the Art and Future Directions. Annu. Rev. Clin. Psychol. 2020; 16: 297–325. PubMed Abstract \| Publisher Full Text\n\nMalone JC, Stein MB, Slavin-Mulford J, et al.: Seeing red: affect modulation and chromatic color responses on the Rorschach. Bull. Menn. Clin. 2013; 77(1): 70–93. PubMed Abstract \| Publisher Full Text\n\nMancuso L, Cavuoti-Cabanillas S, Liloia D, et al.: Tasks activating the default mode network map multiple functional systems. Brain Struct. Funct. 2022; 227(5): 1711–1734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManuello J, Mancuso L, Liloia D, et al.: A co-alteration parceling of the cingulate cortex. Brain Struct. Funct. 2022; 227(5): 1803–1816. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMenon V: Salience Network.Toga AW, editor. Brain Mapping: An Encyclopedic Reference. Amsterdam, NL: Elsevier; 2015; pp. 597–611.\n\nMeyer GJ, Viglione DJ, Mihura JL, et al.: Rorschach performance assessment system. Administration, coding, interpretation, and technical manual. Toledo, OH: Rorschach Performance Assessment System; 2011.\n\nMihura JL, Meyer GJ, Dumitrascu N, et al.: The validity of individual Rorschach variables: systematic reviews and meta-analyses of the comprehensive system. Psychol. Bull. 2013; 139(3): 548–605. PubMed Abstract \| Publisher Full Text\n\nMorawetz C, Bode S, Derntl B, et al.: The effect of strategies, goals and stimulus material on the neural mechanisms of emotion regulation: A meta-analysis of fMRI studies. Neurosci. Biobehav. Rev. 2017; 72: 111–128. PubMed Abstract \| Publisher Full Text\n\nMorris JA, Leclerc CM, Kensinger EA: Effects of valence and divided attention on cognitive reappraisal processes. Soc. Cogn. Affect. Neurosci. 2014; 9(12): 1952–1961. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaito E, Roland PE, Ehrsson HH: I feel my hand moving: a new role of the primary motor cortex in somatic perception of limb movement. Neuron. 2002; 36(5): 979–988. PubMed Abstract \| Publisher Full Text\n\nNickerson LD, Smith SM, Ö ngür D, et al.: Using Dual Regression to Investigate Network Shape and Amplitude in Functional Connectivity Analyses. Front. Neurosci. 2017; 11: 115. Publisher Full Text\n\nNorthoff G: From emotions to consciousness - a neuro-phenomenal and neuro-relational approach. Front. Psychol. 2012; 3: 303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOchsner KN, Silvers JA, Buhle JT: Functional imaging studies of emotion regulation: a synthetic review and evolving model of the cognitive control of emotion. Ann. N. Y. Acad. Sci. 2012; 1251: E1–E24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPace-Schott E, Picchioni D: Neurobiology of dreaming.Kryger M, Roth T, Dement WC, editors. Principles and Practice in Sleep Medicine. Sixth Edition.Amsterdam, NL: Elsevier; 2017.\n\nPanksepp J, Davis K: I fondamenti emotivi della personalità. Un approccio neurobiologico ed evoluzionistico. Milano, ITA: Raffaello Cortina Editore; 2020.\n\nPedrabissi L, Santinello M: STAI. State-Trait Anxiety Inventory - Forma Y. Manuale. Seconda edizione. Firenze, ITA: Giunti Psychometrics; 1989.\n\nPeeters SC, van Bronswijk S , van de Ven V , et al.: Cognitive correlates of frontoparietal network connectivity ‘at rest’ in individuals with differential risk for psychotic disorder. Eur. Neuropsychopharmacol. 2015; 25(11): 1922–1932. PubMed Abstract \| Publisher Full Text\n\nRaichle ME: The brain’s default mode network. Annu. Rev. Neurosci. 2015; 38: 433–447. Publisher Full Text\n\nRaichle ME: The restless brain. Brain Connect. 2011; 1(1): 3–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRazi A, Seghier ML, Zhou Y, et al.: Large-scale DCMs for resting-state fMRI. Network Neuroscience (Cambridge, Mass.). 2017; 1(3): 222–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRedcay E, Warnell KR: A Social-Interactive Neuroscience Approach to Understanding the Developing Brain. Adv. Child Dev. Behav. 2018; 54: 1–44. PubMed Abstract \| Publisher Full Text\n\nRichards HJ, Benson V, Donnelly N, et al.: Exploring the function of selective attention and hypervigilance for threat in anxiety. Clin. Psychol. Rev. 2014; 34(1): 1–13. Publisher Full Text\n\nRijpma MG, Shdo SM, Shany-Ur T, et al.: Salience driven attention is pivotal to understanding others’ intentions. Cogn. Neuropsychol. 2021; 38(1): 88–106. Publisher Full Text\n\nRosazza C, Minati L: Resting-state brain networks: literature review and clinical applications. Neurol. Sci. 2011; 32(5): 773–785. Publisher Full Text\n\nRoy AK, Shehzad Z, Margulies DS, et al.: Functional connectivity of the human amygdala using resting state fMRI. NeuroImage. 2009; 45(2): 614–626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSang L, Qin W, Liu Y, et al.: Resting-state functional connectivity of the vermal and hemispheric subregions of the cerebellum with both the cerebral cortical networks and subcortical structures. Neuroimage. 2012; 61(4): 1213–1225. PubMed Abstract \| Publisher Full Text\n\nSarno I, Preti E, Prunas A: SCL-90-R. Symptom Checklist-90-R. Standardizzazione italiana. Seconda edizione. Firenze, ITA: Giunti Psychometrics; 2011.\n\nSchmidt A, Smieskova R, Simon A, et al.: Abnormal effective connectivity and psychopathological symptoms in the psychosis high-risk state. J. Psychiatry Neurosci. 2014; 39(4): 239–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchutter DJ, van Honk J : The cerebellum on the rise in human emotion. Cerebellum (London, England). 2005; 4(4): 290–294. PubMed Abstract \| Publisher Full Text\n\nSchutter DJ, van Honk J : The cerebellum in emotion regulation: a repetitive transcranial magnetic stimulation study. Cerebellum (London, England). 2009; 8(1): 28–34. PubMed Abstract \| Publisher Full Text\n\nSeeley WW, Menon V, Schatzberg AF, et al.: Dissociable intrinsic connectivity networks for salience processing and executive control. J. Neurosci. 2007; 27(9): 2349–2356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShannon BJ, Dosenbach RA, Su Y, et al.: Morning-evening variation in human brain metabolism and memory circuits. J. Neurophysiol. 2013; 109(5): 1444–1456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith SM, Fox PT, Miller KL, et al.: Correspondence of the brain’s functional architecture during activation and rest. Proc. Natl. Acad. Sci. 2009; 106(31): 13040–13045. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith SM, Jenkinson M, Woolrich MW, et al.: Advances in functional and structural MR image analysis and implementation as FSL. NeuroImage. 2004; 23(Suppl 1): S208–S219. PubMed Abstract \| Publisher Full Text\n\nSporns O: Structure and function of complex brain networks. Dialogues Clin. Neurosci. 2013; 15(3): 247–262. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSridharan D, Levitin DJ, Menon V: A critical role for the right fronto-insular cortex in switching between central-executive and default-mode networks. Proc. Natl. Acad. Sci. 2008; 105(34): 12569–12574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSripada C, Angstadt M, Kessler D, et al.: Volitional regulation of emotions produces distributed alterations in connectivity between visual, attention control, and default networks. NeuroImage. 2014; 89: 110–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevens WD, Buckner RL, Schacter DL: Correlated low-frequency BOLD fluctuations in the resting human brain are modulated by recent experience in category-preferential visual regions. Cerebral Cortex (New York, N.Y.: 1991). 2010; 20(8): 1997–2006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStippich C, Ochmann H, Sartor K: Somatotopic mapping of the human primary sensorimotor cortex during motor imagery and motor execution by functional magnetic resonance imaging. Neurosci. Lett. 2002; 331(1): 50–54. PubMed Abstract \| Publisher Full Text\n\nStoodley CJ, Schmahmann JD: Functional topography in the human cerebellum: a meta-analysis of neuroimaging studies. NeuroImage. 2009; 44(2): 489–501. PubMed Abstract \| Publisher Full Text\n\nStoodley CJ, Schmahmann JD: Functional topography of the human cerebellum. Handb. Clin. Neurol. 2018; 154: 59–70. Publisher Full Text\n\nStoodley CJ, Valera EM, Schmahmann JD: Functional topography of the cerebellum for motor and cognitive tasks: an fMRI study. NeuroImage. 2012; 59(2): 1560–1570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzucs D, Ioannidis JP: Sample size evolution in neuroimaging research: An evaluation of highly-cited studies (1990-2012) and of latest practices (2017-2018) in high-impact journals. Neuroimage. 2020; 221: 117164. PubMed Abstract \| Publisher Full Text\n\nTang S, Wang Y, Liu Y, et al.: Large-scale network dysfunction in α-Synucleinopathy: A meta-analysis of resting-state functional connectivity. EBioMedicine. 2022; 77: 103915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeeuw J, Pol HEH, Boomsma DI, et al.: Reliability modelling of resting-state functional connectivity. NeuroImage. 2021; 231: 117842. Publisher Full Text\n\nTenenhaus M: La régression PLS: théorie et pratique. Pairs, FRA: Editions Technip; 1998.\n\nTurner BM, Paradiso S, Marvel CL, et al.: The cerebellum and emotional experience. Neuropsychologia. 2007; 45(6): 1331–1341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVecchio A, Roberti E, Pulido SG, et al.: Only an Inkblot? A Literature Review of the Neural Correlates of the Rorschach Inkblot Test. Neurosci. Biobehav. Rev. 2023; 152: 105281. PubMed Abstract \| Publisher Full Text\n\nTurken AU, Dronkers NF: The neural architecture of the language comprehension network: converging evidence from lesion and connectivity analyses. Front. Syst. Neurosci. 2011; 5: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVitolo E, Diano M, Giromini L, et al.: Markers of emotion regulation processes: A neuroimaging and behavioral study of reappraising abilities. Biol. Psychol. 2022; 171: 108349. PubMed Abstract \| Publisher Full Text\n\nVitolo E, Giromini L, Viglione DJ, et al.: Complexity and cognitive engagement in the rorschach task: an fMRI study. J. Pers. Assess. 2021; 103(5): 634–644. PubMed Abstract \| Publisher Full Text\n\nWard JH: Hierarchical grouping to optimize an objective function. J. Am. Stat. Assoc. 1963; 58(301): 236–244. Publisher Full Text\n\nWoolrich MW, Jbabdi S, Patenaude B, et al.: Bayesian analysis of neuroimaging data in FSL. NeuroImage. 2009; 45(1 Suppl): S173–S186. PubMed Abstract \| Publisher Full Text\n\nYeshurun Y, Nguyen M, Hasson U: The default mode network: where the idiosyncratic self meets the shared social world. Nat. Rev. Neurosci. 2021; 22(3): 181–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZuo XN, Kelly C, Adelstein JS, et al.: Reliable intrinsic connectivity networks: test-retest evaluation using ICA and dual regression approach. NeuroImage. 2010; 49(3): 2163–2177. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "308743", "date": "12 Aug 2024", "name": "Filippo Aschieri", "expertise": [ "Reviewer Expertise Personality assessment", "Rorschach test" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript could represent a model of research methodology for the study of the Rorschach test with the fMRI. As the authors correctly note, it could complement findings of research that used a different approach (see \"Despite the relevance...\" section). I find the study complex and have to disclaim that I lack the competence to assess the appropriateness of the analyses of neurophysiological data. I will limit my observations to the Rorschach.\n1) Introduction. Despite being well-written, it lacks updated references to the Rorschach test. Add recent literature about the “nature” of theRorschach: [Ref:1,2,3](the latter is relevant also to the concept of projection!). Also [Ref:4] should be mentioned when discussing how the 10 cards work. You need to add a paragraph about the limits of the CS, and the variables that either lack of research or that lack of validity [Ref:5]. Relatedly, I would select only valid variables for the analyses, adding Complexity which is too much of a relevant variable for the protocol interpretation Ref:[6]. If you cannot add Complexity to the analyses at this stage, and want to use CS variables that have questionable validity, this must be mentioned when discussing the corresponding results, and in the Limits section.\n2) Section 1.2. - \"Despite...\" --> Incomplete sentence: - \"However\"--> Why however? -\"Nowadays, the more studied more because\"--> Broken sentence -\"The activity of the DMN has also been..\"--> Why adversative? -\"If the DMN..\" --> why future verb tense? \"It complies...\"-->It includes? -\"The question, therefore..\"--> Since you report a reasonable answer to the question, it seems a rhetorical one, which should be avoided. -\"Another resting-state..\"--> repetition of the word \"network\" -\"are in line\"--> agree -\"Stevens et al. (2010)\"--> sorry, unclear what Stevens et al suggest! -From \"The executive...\" and onwards--> these last paragraphs are comparatively less elaborated than the previous ones, maintain consistency. - Check that all areas considered in the analyses are described and introduced here. I imagine there is not so much literature, but these descriptions hardly cover any relevant aspect of personality functioning. This is the focus of the article, so make an effort and focus the information on which area is responsible for which personality function you aim to measure with the Rorschach.\n3) Section 1.3 - \"ancestral\" --> correct term? -\"Based on the literature presented so far, it is possible to ask whether neuroimaging techniques can identify the neural correlates..\"--> this question makes sense as long as you convincingly argue that the DMN is connected to personality: which is never mentioned in the previous section. I would re-work the section titled 1.2 Resting State Large Brain Network making sure that the components you mention are connected with “personality” aspects of human functioning.\n4) Section 2. -\"rest with their eyes open and to passively start at a point\"--> were eyes closed or open? check the inconsistency in the 2.4.1 section, first line; Stare instead of Start?\n5) Section 2.2, Rorschach -\"resigned\"--> recoded -\"Color-Shape\"--> \"color-form\" - Not sure how convincing is the selection of CS variables. I would only focus on the variables that are conceptually relevant and connected to the DMN. How were these variables selected? It seems you are also using R-PAS variables (Critcont), which is ok, but needs to be acknowledged. Complexity is missing. Also, there are redundancies among the selected variables (Xy and An are also part of Critcont), some variables lack empirical support (D, AdjD, CDI). Make sure you explain why you choose these variables and for those without validity, list the problem among the limits of the article. Also, you need to stress that 24 variable for 24 subjects make the power of the analysis very low. This is another substantial limit of the article. I realize this is an explorative article, so it might be less fatal than otherwise. However, the exploratory nature of the study should be reflected in the introduction, in the title, and in the limits of the manuscript.\n\n6) Section 3.1 - as a newby to PLS regresssion I wonder how significant Factors 2 and 3 are, and what is their interpretative meaning. I would add here a sentence describing the interpretation of these results. Also, it seems that each IC explains a percentage of variance, I cannot understand how the total variance is computed (e.g., VS+DMN+SMOT alone account for more than 100% ov the variance)\". -figure 12--> Make sure that the dendrogram is also interpretable. No information is provided in this section and readers need to wait until the discussion to decipher the meaning authors give to these data. I strongly recommend to provide immediately the inferences of these results. First cluster is reality testing. Second cluster: distress, but why FQ-? and why r? and how come Critcont is so far apart from Xy?Third cluster: seems close to complexity. Fourth cluster: it seems related to “others” and relationships, but An and Critcont?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-803
https://f1000research.com/articles/13-800/v1	16 Jul 24	{ "type": "Research Article", "title": "Evaluation of endophytic colonization and establishment of entomopathogenic fungi against Tuta absoluta (Lepidoptera: Gelechiidae) in tomato plants", "authors": [ "Dereje Geremew", "Tadale Shiberu", "Ararsa Leta", "Tadale Shiberu", "Ararsa Leta" ], "abstract": "Background The tomato, Solanum lycopersicum L., is one of the most important horticultural crops that can be consumed fresh or after being processed worldwide. The tomato leaf miner (Tuta absoluta) is one of the most devastating pest to tomato plants due to its mine-feeding nature in the mesophyll tissue of the plant. Fungal entomopathogens can exist naturally in plants as an asymptote. This study aimed to detect the endophytic colonization of Beauveria bassiana and Metarhizium robertsii within tomato plants via artificial inoculation and their virulence effects on Tuta absoluta.\n\nMethods Isolates with the highest percent germination and virulence against T. absoluta were selected for endophytic evaluation within tomato plants by different artificial inoculation techniques.\n\nResults This study revealed that, isolates with the highest percent germination and virulent to Tuta absoluta had the potential to colonize tomato plants. The result showed that, the maximum mortality rate (97.5%) of Tuta absoluta larvae was achieved by Metarhizium robertsii isolate K-61 at a concentration of 1x108conidial/ml at 7 days post inoculated. However, the highest cumulative mortality (100%) was recorded by Beauveria bassiana isolate APPRC-27 at 10 days post inoculated through the direct contact method. The highest endophytic colonization was registered by isolate APPRC-27 (76.67%) at 7 days post-inoculated using the leaf spray technique, but it declined to 11.67% after 28 days of inoculated. In the case of the seedling inoculation technique, the highest endophytic colonization was obtained in the root tissues of tomatoes at 28 days of inoculated by isolate K-61.\n\nConclusions This study revealed that the leaf spray inoculation technique was the most effective method, followed by seedling inoculation, for the deployment of Beauveria bassiana and Metarhizium robertsii endophytes in tomato plant tissues. Therefore, virulent Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of Tuta absoluta if deployed as endophytes.", "keywords": [ "Beauveria bassiana", "Colonization", "Endophytic", "entomopathogenic fungi", "Metarhizium robertsii", "Tuta absoluta" ], "content": "Introduction\n\nThe tomato, Solanum lycopersicum L., is one of the most cultivated vegetables worldwide due to its low-fat content and excellent source of dietary fiber, minerals, vitamins, and antioxidants (Erdinc et al., 2018). It can be consumed fresh and/or processed into a wide variety of manufactured products (Sousa et al., 2008). World-wide tomato cultivation suffers enormous losses due to insect attacks, which reduce the quality of the fruits and spread plant pathogens. The South American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is native to South America and one of the primary global pests of tomatoes that causes 80–100% yield losses (Desneux et al., 2010). The pest first arrived in Ethiopia in 2012 via Sudan and later expanded to other parts of the country (Mansour et al., 2018; Shiberu & Getu, 2017; Gashawbeza and Abiy, 2013).\n\nIn Ethiopia, the yield loss due to T. absoluta was reported in the range of 60.08% to 82.31% (Shiberu & Getu, 2018). It also reduces the quality of the yield, which directly affects marketability, and causes secondary infection by creating wounds. This increases the cost of production to farmers, which they pay for pesticides in the country, particularly in West Shewa. Considering alternative ways of protecting EPF from adverse conditions, the incorporation of EPF in plants as endophytes would appear to be a highly interesting and promising approach (Vega, 2018).\n\nThe endophytes contribute to plant protection by controlling herbivores, insects, and pathogens on the host plant. In addition, they may strengthen plant resistance to pathogens and biotic and abiotic stresses (Ahlholm et al., 2002; Kogel et al., 2006). As a result of direct contact with potential insect hosts, entomopathogenic fungi (EPF) infect their hosts by penetrating through cuticular exoskeletons (Zimmermann, 2007).\n\nEndophytic microorganisms are now attracting great interest from researchers as an alternative source for the control of plant pests. The introduction of entomopathogenic fungi as endophytes could protect the pathogen from adverse environmental conditions, reducing the negative effects of ultraviolet radiation and the limitations of low humidity on development. Entomopathogenic fungi could be deployed as a biological control strategy against specific pests, especially those with life cycles that require feeding inside leaves, stems, rhizomes, roots, and seeds, behaviors that reduce their exposure to synthetic insecticides and other control methods (Resquín-Romero et al., 2016).\n\nCurrently, EPF is more promising than chemical pesticides for the control of pests in Ethiopia due to pesticide resistance. Colonization of a host plant by an endophyte is influenced by the inoculation method, species of fungal endophytes, and the host plant species itself. Based on the inoculation technique, the endophytes differ in their ability to colonize different plant parts and persist over a crop growth cycle (Akello et al., 2007; Brownbridge et al., 2012). Knowing the endophytic association and establishment of EPF in tomato plants helps to modify the inoculation techniques for plant parts based on the behavior of targeted pests, particularly T. absoluta, which lives in the mesophyll tissue of the tomato plant. However, such successful work is not available in Ethiopia on endophytic colonization of EPF on tomato plants against T. absoluta. Therefore, this study was conducted to detect endophytic colonization of B. bassiana and M. robertsii with tomato plants by different inoculation techniques and their virulence effect against T. absoluta.\n\n\nMethods\n\nThe experiment was conducted at the Ambo Agriculture Research Center under laboratory and glasshouse conditions. Ambo is far away from Addis Ababa, 110 km to the west, at a geographical coordinate of 8°59’ N latitude and 37.85°E longitude, with an altitude of 2100 meters above sea level. Daily temperature ranges between 15°C-29°C and annual 22°C on average.\n\nConidia were obtained from pure cultures grown on the 65g of Sabouraud Dextrose Agar (SDA) Microxpress® REF: 201190030500 which were incubated for 14 days at 27±1°C under dark conditions. Conidia were harvested by disposable cell scrapers and added into test tubes containing tween 80 (0.001%v/v). Suspensions were vortexed well for 2 min and adjusted to 1 × 108 conidia/ml using a Neubauer haemocyutometer (Gurulingappa et al., 2010; Hassan et al., 2019; Ozdemir et al., 2021).\n\nThe mass rearing of T. absoluta was established in 2021 from larvae collected from tomato plants without any history of pesticides in fields surrounding Ambo areas. The Roman VF variety tomato plant was grown in the glasshouse to maintain the life cycle of T. absoluta. The host plant, the tomato, was maintained under quarantine to discard the presence of potential diseases and parasites. Groups of four cages (height: 30 cm; diameter: 23 cm) were used for rearing, each with one tomato plant. The first cage was used for adult oviposition, the second for 1st to 2nd instar larvae, the third for 3rd to 4th instars, and the fourth cage for maintaining the pupae and emerging adults. Larvae were fed pesticide-free tomato seedlings (S. Lycopersicum cv. Roman VF), whereas adults were fed a 15% honey-water solution (Allegrucci et al., 2017). The maintenance of the insects and mass rearing were conducted under glasshouse conditions at Ambo Agricultural Research Center (AmARC).\n\nHigh-virulent B. bassiana and M. robertsii isolates that were prescreened on G. melonella were again tested against 2nd larval instars of T. absoluta (Sabbour and Singer, 2014). Five B. bassiana and three M. robertsii isolates were tested at a concentration of 1 × 108 conidial/ml for the screening of virulence against 2nd instar larvae of T. absoluta. Briefly, spore suspensions were adjusted to 1 × 108 conidia/ml using Tween 80 (0.001%v/v) in sterile distilled water.\n\nTomato leaves Surface disinfection was done by washing with 1% sodium hypochlorite for 2 min, 2 min in ethanol (70%), and rinsing 3 times with sterile distilled water. Surface-sterilized leaves were placed on sterile plastic plates, sprayed with 3 ml of fungal spores, and air-dried under the safety cabinet for 3 minutes. Ten second-instar larvae of T. absoluta were separately released over leaves and incubated for 7 days at room temperature. A control treatment was sprayed with Tween 80 (0.001%) and incubated under the same conditions.\n\nThe mortality of larvae was recorded daily, and dead larvae were surface sterilized and placed on sterile plastic plates containing moistened filter paper. Plates with larval cadavers were moistened daily with distilled sterile water to enhance mycosis development over the cadavers and incubated at room temperature.\n\nMycosis percentages were calculated using the percentage of cadavers showing external fungal growth out of the total number of tested insects (Sabbour, 2014). The experiment was conducted under laboratory conditions in the CRD with nine treatments and replicated four times.\n\nTo determine the infectivity of the fungal isolates against T. absoluta, four isolates were evaluated by the direct contact method (Allegrucci et al., 2017). Three M. robertsii isolates, namely K-63, K-61, and K-102, with one B. bassiana APPRC-27, were selected based on their conidial viability, yield, production, and radial growth for their virulence against T. absoluta, according to Dotaona et al. (2015), Campos-Herrera et al. (2021), and Gebremariam et al. (2021).\n\nSurface disinfected 400 tomato leaf discs of approximately 2 cm diameter were cut, and 320 of them were immersed 0.001% in solution of Tween 80 of B. bassiana and M. robertsii (1 × 108 conidia/ml) separately, while the other 80 (controls) were immersed in a 0.001% conidia-free solution of Tween 80. The experiment was conducted in the laboratory by CRD with five treatments and replicated four times, with each treatment containing 20 2nd instar larvae. Leaf discs were placed on a plastic plate separately, with a second instar larva per disc. Every 24 hours, larval mortality was recorded. Larval mortality was evaluated daily for 10 consecutive days. Dead larvae were removed and the surface disinfected.\n\nData on mortality were corrected for their control mortality by using Abbot’s (Abbott, 1925) formula:\n\nWhere: CM=Corrected mortality, C, mortality in the untreated larvae, and T is mortality in the treated larvae.\n\nThe cadavers were placed in sterile Petri dishes with filter paper moistened with sterile distilled water, and mycosis was confirmed under a stereomicroscope by microscopical examination of the dead insects (Allegrucci et al., 2017).\n\nTomato seed surface sterilization\n\nThe Roman VF variety was brought from Melkassa ARC and sown in the glasshouse at AmARC. Before sowing, the seeds were surface sterilized by successive immersion in 1% sodium hypochlorite for 2 minutes, then 70% alcohol for 3 minutes, and finally three washes in sterile distilled water (Brownbridge et al., 2012). Seeds were then placed on sterile filter paper to dry for 30 minutes and divided into two portions. One portion was used for seedling inoculation techniques, and the other was used for leaf spraying and root dipping after seedling emergence.\n\nGrowing the tomato plants\n\nSeeds were planted for each inoculation technique separately, and pots were filled with compost, loam soil, and sandy soil in the ratio of 1:1:2, respectively.\n\nThe soil was sterilized in an autoclave for 6 hours at 121°C before use at Ambo University’s Mamo Mezmir campus. The experiment was conducted under glasshouse conditions at 25–30°C, and all necessary agronomic practices were followed for the growth of tomato plants. Treatments were arranged in CRD and replicated four times for each inoculation technique. A total of 576 tomato seeds were sown, and all agronomic practices were done as recommended.\n\nThe most virulent isolates of B. bassiana and M. robertsii against T. absoluta were selected for the examination of endophytic colonization in tomato plants. Two M. robertsii and one B. bassiana isolate were tested with three inoculation techniques (leaf spray, root dipping, and seedling inoculation) for the presence or absence of endophytic colonization of these fungi in tomato plant tissues. Endophytic colonization and establishment potential of B. bassiana and M. robertsii isolates were evaluated by introducing them into tomato plants through seedling inoculation, root dipping, and leaf spraying techniques (Allegrucci et al., 2017; Russo et al., 2019; Silva et al., 2020). A total of 480 tomato plants were randomly chosen and used for the detection of endophytic colonization.\n\nLeaf spraying\n\nThe leaves of tomato plants (7 weeks old) were sprayed with three ml of 1 × 108 conidia ml–1 suspension in a glass hand sprayer (30 ml capacity). To avoid conidial runoff into the soil, the top of each pot was covered with aluminum foil (Posada et al., 2007). Control plants were sprayed with a conidia-free solution of 0.001% Tween 80 (Saragih, 2019; Sinno et al., 2021).\n\nSeedling inoculation\n\nEndophytic colonization of tomato seedlings with isolates was conducted by inoculating seedlings with B. bassiana and M. robertsii conidia. The seeds were surface disinfected in 1% sodium hypochlorite for 2 minutes, followed by 70% ethanol for 2 minutes, and rinsed 3 times with sterile distilled water (Arnold and Herre, 2003; Posada and Vega, 2006). Surface-sterilized seeds were placed in plastic Petri dishes lined with filter paper, moistened with 1 ml sterilized distilled water, and placed at 70% relative humidity for 6 days to stimulate germination. B. bassiana and M. robertsii Conidial suspension was added to germinated seeds at 1 × 108 conidia/ml with tween 80 (0.001%v/v). Two days post-inoculation of fungus, the seedlings were transferred to pots filled with sterilized soil and planted carefully without damaging the roots. For control treatment, surface sterilization was conducted as mentioned above, and control seeds were treated with tween 80 (0.001%v/v) 6 days post-germination (Silva et al., 2020).\n\nRoot dipping\n\nFor root dipping, three weeks of seedlings were used after emergence. Each seedling was removed from the pot and rinsed three times with sterile distilled water. The ends of the roots were cut for better absorption and placed individually in test tubes with 2 ml of a conidial suspension containing 1 × 108 conidia/ml (Akello et al., 2007; Bamisile et al., 2018; Allegrucci et al., 2020). Each tube was covered with aluminum foil to prevent damage from UV rays. The roots of control plants were submerged in sterile distilled water. Control and treated plants were incubated at 26°C with 70% relative humidity and under a 12:12 (L:D) photoperiod for 24 h. Thereafter, both the control and treated plants were placed on sterile filter paper until completely dried and then replanted in the same pots. Seedlings in all treatments were watered as needed and maintained in a glasshouse.\n\nFor each inoculation technique, Colonization of tomato plants by B. bassiana and M. robertsii was assessed at 7, 14, 21, and 28 DPI. The evaluation was conducted for the presence or absence of B. bassiana and M. robertsii conidia on 480 plants in total, 120 plants for each inoculation technique, and 40 plants for each isolate (360 treated plants and 120 control plants). Ten plants were randomly chosen at each re-isolation time and taken to a laboratory for the detection of endophytic colonization of isolates through plant tissues. Ten replications were made for each plant part (leaf, stem, and root), and 30 replications were used for each isolate.\n\nAccordingly, tomato plants were surface disinfected as stated above, and complete disinfection of leaves was checked by plating 100 ml of the last rinsing water of each sample onto SDA (Gurulingappa et al., 2010). Plant parts were dried on sterile filter paper under a laminar flow cabinet, and their edges were cut to remove dead tissue due to the sterilization process.\n\nExamination of conidia was conducted by cutting plant parts (leaf, stem, and root) into six small pieces, approximately 5 mm2 for leaf and 5mm for stem and root, by using surgical blades; 2880 pieces were used in total, then placed on a Petri dish containing SDA with antibiotic (Vega et al., 2008a).\n\nConfirmation was conducted by visual observation under a stereomicroscope for the presence or absence of fungi after 10 days of incubation of the culture at 27 °C. For each inoculation technique, the colonization percentage was calculated as follows (Greenfield et al., 2016).\n\nData on larval mortality was calculated using Abbot’s formula (1925). Means were separated using Tukey’s Honestly Significant Difference (HSD) at P 0.05 for screening against T. absoluta and evaluation of isolates. The mean colonization (expressed in percentage) data were arcsine transformed to stabilize the efficacy of the analysis of variance (Gomez and Gomez, 1984).\n\n\nResults and Discussions\n\nScreening the virulence of isolates against T. absoluta larvae\n\nThe result revealed that there is a highly significant difference among isolates in the mean percent mortality of T. absoluta larvae (F = 67.89; DF = 8; P < 0.0001). Isolate K-61 showed the highest mortality rate (97.5%) against the larval stage of T. absoluta under laboratory conditions after 7 days of inoculation (Table 1). The lowest mortality rate was registered by APPRC-44BC and K-5 isolates (72.5%).\n\nFungal mycosis was developed on the dead cadavers of T. absoluta larvae, which were able to select the most virulent isolates against T. absoluta (Figure 1). There is a highly significant difference among treatments in mycosis percentage on dead cadavers, and no mycosis was observed in the control treatment (Table 1).\n\nA = B. bassiana, B = M. robertsii.\n\nA higher mycosis percentage was obtained from K-61 and K-91, with 67.5% and 65%, respectively. The minimum mycosis percentage was recorded by K-5 (20%), followed by APPRC-44BC with 25% on cadavers of T. absoluta larvae.\n\nThis result showed that all isolates were highly virulent to the larval stage of T. absoluta under laboratory conditions at 1 × 108 conidial/ml after 10 days of inoculation. The highest cumulative mortality (100%) was obtained from B. bassiana isolate APPRC-27 (Figure 2). According to the present study, B. bassiana and M. robertsii were able to infect the larval stage of T. absoluta by direct contact method.\n\nMedian lethal time (LT50)\n\nThis study revealed that fungal isolates with the highest mortality rate against T. absoluta record the lowest LT50 days, and vice versa. As a result, the lowest median lethal time (LT50) and (LT90) of T. absoluta larvae were attained by B. bassiana isolate APPRC-27 with 3.67 and 6.19 days, respectively, at 1 × 108 conidia/ml (Table 2).\n\nLeaf spray\n\nThe result showed that a higher percentage of colonization was obtained at 7 DPI by B. bassiana isolate APPRC-27 with 76.67%, 35.00%, and 16.67% in leaves, stems, and roots, respectively, through the leaf spray method of inoculation (Table 3)\n\nThe lowest endophytic colonization was recorded after 28-day inoculation with 11.67% (SE±3.56) in leaves and 1.67% (SE±1.67) stems; no B. bassiana was detected in tomato roots at 28 DPI.\n\nIn the case of M. robertsii, successful endophytic colonization of tomato plants was obtained by the leaf spray method with 53.33% at 7-DPI in leaves (Figure 3), and no fungal colonization occurred in root tissues after 28 days of inoculation. This suggested that the recovery of B. bassiana and M. robertsii from inoculated hosts decreased from the site of inoculation in plant tissue over time.\n\nA = colony color (front) of B. bassiana, B = colony color (front) M. robertsii.\n\nSeedling inoculation\n\nIn contrast to the leaf spray technique, this study showed tomato plants were successfully colonized by B. bassiana isolate APPRC-27 28 days post inoculated, with 65% of colonization in roots by the seedling inoculation technique (Table 4). The highest endophytic percent of colonization was detected from tomato roots, followed by stems, in this method after 28 days of inoculated by all isolates.\n\nMaximum colonization mean percent (71.67% SE±0.98) was obtained from M. robertsii isolate K-61 at 28 DPI from tomato roots, whereas minimum colonization was obtained from M. robertsii isolate K-102 (8.33%±SE ±0.22) in leaf tissue at 7 DPI (Figure 4).\n\nC = colony color of isolate APPRC-27 from tomato roots; C (2) colony color of isolate APPRC-27 from tomato leaves; D = colony color (front-to-left, back-to-right) of isolate K-61.\n\nThis result estimated that the persistence of B. bassiana and M. robertsii in the tomato plants increased up to 28 days after inoculation by the seed inoculation technique.\n\nIn all techniques of inoculation, there was a variation in endophytic colonization of tomato plants by M. robertsii and B. bassiana over time, and there was no fungal detection from uninoculated tomato plants. From the results, we can suggest that inoculation techniques greatly affect the movement and persistence of fungal isolates through the plant tissues of tomatoes. There is a significant difference in the interaction of inoculation techniques for the establishment of fungal isolates as endophytes within plant tissues. Accordingly, the leaf spray inoculation technique was a successful method for the endophytic colonization of entomopathogenic fungi in tomato plants, which were used against tomato leaf miner (T. absoluta). This study suggested that the establishment of M. robertsii and one B. bassiana as endophytes in the tomato plant reduces the population of T. absoluta by interfering with the feeding ability of larvae.\n\nRoot dipping technique\n\nIn the case of the root dipping technique of inoculation, the highest endophytic colonization of tomato plants was recorded by M. robertsii isolate K-61 after 7 days (43.33%) in root tissues, and it declined to 10% at 28 DPI. The Lowest endophytic colonization (3.33%) was registered in tomato plants treated with B. bassiana isolate APPRC-27 after 28 days of inoculation in root tissues, and no fungal colonization was detected in stems and leaves on the same day (Table 4).\n\nThe result of interaction between two variables (inoculation techniques and days DPI at 7, and 28) showed that there is a highly significant difference in endophytic colonization of tomato leaves by fungal isolates (DF = 3, F = 1001.80, P < 0.0001). As a result, the leaf spray technique shows an increase in endophytic colonization by entomopathogenic fungi up to 7 DPI (38.30%) and a decline to 14.86% at 28 days in the leaves of the tomato plant. However, Seedling inoculation technique increased over time and higher endophytic colonization of entomopathogenic fungi obtained at 28 DPI (27.61%) (Figure 4).\n\nIn the case of root colonization of tomato plants by entomopathogenic fungi, the interactions of two variables show that the seedling inoculation technique shows a maximum endophytic colonization at 28 DPI.\n\nIn this study, selected isolates were screened for their pathogenicity against T. absoluta larvae. Pathogenicity tests revealed that all isolates showed 72.5% up to 97.5% mean mortality of T. absoluta larvae at a concentration of 1 × 108 conidial/ml after 7 days of inoculation. This result agrees with the study conducted by Ayele et al. (2020), where maximum mortality rates (95%) were registered on the 2nd larval instar of T. absoluta by M. robertsii isolate AAUM39 at a concentration of 1 × 107 conidial/ml 7 days post-inoculation. Shibiru and Getu (2017) also reported that the mortalities caused by B. bassiana isolate at the different concentrations ranged from 79.17% to 95.83% under laboratory conditions, and 87.50% mortality was registered by M. robertsii after 7 days of treatment application at 2.5 × 109 conidia/ml.\n\nThe entomopathogenic B. bassiana fungus could cause larval mortality of up to 95% compared to chemical treatment of 88% (El Kichaoui et al., 2016). In this study, certain selected isolates were evaluated for their virulence against T. absoluta larvae by direct contact. B. bassiana caused the highest cumulative mortality of larvae (100%) at 10 DPI at a concentration of 1x108 conidia/ml. A similar study indicated that direct contact by leaf spraying showed a higher mortality rate and a lower MST value than indirect contact of T. absoluta larvae with conidia (Allegrucci et al., 2017).\n\nBy introducing entomopathogenic fungi as endophytes, the pathogen could be shielded from unfavorable environmental factors, lessening the impact of UV radiation and the developmental constraints of low humidity (Resquín-Romero et al., 2016). Vega et al. (2008b) reported that 90 genera of entomopathogenic fungi are known, and most of them exist as endophytes in plants, but only 12 species have been tested as biocontrol agents.\n\nIn the present work, B. bassiana and M. robertsii were successfully colonized tomato plants endophytically by using different artificial inoculation techniques. The highest endophytic colonization was attained by B. bassiana isolate APPRC-27 (76.67%) through leaf spray technique, followed by M. robertsii isolate K-61 (71.67%) by seedling inoculation technique at 7 and 28 DPI, respectively. This revealed that the colonization of B. bassiana observed in tomato leaves at 7-DPI was 29.44% by the leaf spray method, which was reported as the most effective method of inoculation for the establishment of entomopathogenic fungi as an endophyte (Allegrucci et al., 2017). This may be due to the stomata structures, which are the main entry and channel for many pathogenic fungi (Shikano et al., 2017; Gonzalez et al., 2021). However, B. bassina may have the potential to invade plant tissues if stomata are encountered under unfavorable conditions (Wagner & Lewis, 2000).\n\nThis study revealed that endophytic colonization of fungal conidia in tomato plants was increased over time up to 30 DPI by using the seedling inoculation technique, and the highest endophytic colonization was detected from tomato root tissues after 28 days of inoculation. The result is consistent with the root tissues and stems displayed the highest percent of colonization at 7 DPI, with 80%, and at 30 DPI, all samples from all parts of the tomato plants were 100% positive for fungal colonization of B. bassiana isolate LPP139 by the seedling inoculation method, which results in relatively rapid and lasting colonization of tomato plants (Silva et al., 2020).\n\nHowever, endophytic colonization of tomato plants by fungal conidia decreased over time by using leaf spray and root dipping techniques of inoculation for both species of tested isolates. In the case of root dipping, the highest recovery of endophytic fungi was obtained by M. robertsii isolate K-61 (43.33%) after 7 days of inoculation. Similarly, successful endophytic colonization of tomato plants was achieved through the root dipping method with 12.22% mean values in root tissues at 7 DPI by B. bassiana (Allegrucci et al., 2017).\n\nThe result is consistent with the fact that Metarhizhium species prefer root tissues to leaves as endophytic colonization after artificial inoculation, while B. bassiana prefers aerial plant parts (Behie et al., 2015). M. robertsii has a well-established relationship with the rhizosphere (Leger, 2008; Bruck, 2005). However, successful endophytic colonization of B. bassiana occurred in tomato root tissue at 37.75% at 7 dpi by the root dipping method (Mwamburi, 2021).\n\nIn all inoculation techniques, this study revealed that B. bassiana and M. robertsii can migrate endophytically from the inoculated sites and colonize other plant tissues in the tomato plant. This supports the systematic translocation and colonization of fungal inoculums in plant tissues from sites inoculated to other plant tissues (Tefera and Vidal, 2009; Sánchez-Rodríguez et al., 2015; Omukoko, 2018; Sinno et al., 2021). Elena et al. (2011) also reported that M. robertsii was capable of moving within the plant and B. bassiana was able to colonize corn and maize within plant tissues by stem injection technique.\n\n\nConclusions\n\nT. absoluta is a major pest of tomato plants and difficult to control due to its mine-feeding nature in mesophyll tissues. As a result, using entomopathogenic fungi such as B. bassiana and M. robertsii as endophytes is a novel and eco-friendly method for the management of tomato leaf miner due to its migratory potential, deployment, and long persistence over chemical insecticides.\n\nIsolates with high germination percent and maximum spore production were highly virulent to 2nd-instar larvae of T. absoluta. Accordingly, B. bassiana isolate APPRC-27 was highly virulent to T. absoluta by direct contact method with 100% cumulative mortality, followed by M. robertsii isolate K-61 (96.49%) at 10-days post-inoculated at a concentration of 1 × 108 conidial/ml. Among the tested isolates, the highest colonization percent was achieved by B. bassiana isolate APPRC-27 (76.67%) from tomato leaves at 7 days post-inoculation through the leaf spray technique.\n\nThis study confirmed that the virulent isolates had the potential to colonize and migrate in tomato plant tissues. However, endophytic colonization reduced over time at 28 days post-inoculation for all isolates by leaf spray and root dipping techniques while increasing by seedling inoculation techniques. The inoculation techniques greatly affect the persistence and establishment of B. bassiana and M. robertsii as endophytes in tomato plant tissues over time.\n\nUsing two M. robertsii isolates, K-61 and K-102, and a B. bassiana isolate, APPRC-27, is the best biological option against larvae of T. absoluta. The leaf spray and seedling inoculation techniques are the most effective with B. bassiana isolate APPRC-27 and M. robertsii isolate K-61, respectively, for the establishment of fungal conidia as endophytes in the tomato plant tissues against T. absoluta.\n\nHowever, further studies must be conducted on the persistence and mechanism of migration of B. bassiana and M. robertsii in tomato plant tissues concerning physiological activities within the tomato plant. Moreover, further studies are needed on the factors attributed to the efficacy and endophytic association of B. bassiana and M. robertsii in tomato and other Solanaceae plants against T. absoluta under field conditions. The compatibility of virulent isolates with other management options also needs to be taken into consideration while developing integrated pest management strategies for the control of T. absoluta.", "appendix": "Data availability\n\nFigshare: Arcsine transformed data, evaluation of inoculation techniques of endophytic entomopathogenic fungi, lt50% https://doi.org/10.6084/m9.figshare.25585323.v1 (Geremew, 2024).\n\nThis project contains the following underlying data:\n\n• Arcsine transformed data\n\n• evaluation of inoculation techniques of endophytic entomopathogenic fungi\n\n• LT50%\n\nData are available under the terms of https://creativecommons.org/licenses/by/4.0/ (CC BY 4.0 DEED).\n\n\nAcknowledgments\n\nWe are grateful for the financial support provided by the Ethiopian Institute of Agricultural Research (EIAR). The biocontrol research team at the Ambo Agricultural Research Center is appreciated by the authors for helping with the laboratory trials. The authors also acknowledge the help with the laboratory work provided by Mr. Tesfaye Abdisa, Ms. Aberash Chala, and Mr. Denberu Kebede. Additionally, the authors acknowledge Tariku Hunduma (PhD) for his technical assistance in developing the technique. The authors attested to the fact that the EIAR and the people listed in this section gave permission for their names to be published in this work.\n\n\nReferences\n\nAbbott WSJJE: A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925; 18(2): 265–267. Publisher Full Text\n\nAhlholm JU, Helander M, Henriksson J, et al.: Environmental conditions and host genotype direct genetic diversity of Venturia ditricha, a fungal endophyte of birch trees. Evolution. 2002; 56(8): 1566–1573. PubMed Abstract\n\nAkello J, Dubois T, Coyne D, et al.: Colonization and persistence of the entomopathogenic fungus, Beauveria bassiana, in tissue culture of banana. 8th African Crop Science Society Conference, El-Minia, Egypt, 27-31 October 2007. African Crop Science Society. 2007; pp. 857–861.\n\nAllegrucci N, Velazquez M-S, Russo M-L, et al.: Establishment of the entomopathogenic fungus Beauveria bassiana as an endophyte in Capsicum annuum and its effects on the aphid pest Myzus persicae (Homoptera: Aphididae).2020; 68(4): 1084–1094.\n\nAllegrucci N, Velazquez MS, Russo ML, et al.: Endophytic colonisation of tomato by the entomopathogenic fungus Beauveria bassiana: The use of different inoculation techniques and their effects on the tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae). J. Plant Prot. Res. 2017; 57: 331–337.\n\nArnold AE, Herre EA: Canopy cover and leaf age affect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia. 2003; 95(3): 388–398. PubMed Abstract \| Publisher Full Text\n\nAyele BA, Muleta D, Venegas J, et al.: Morphological, molecular, and pathogenicity characteristics of the native isolates of Metarhizium anisopliae against the tomato leafminer, Tuta absoluta (Meyrick 1917)(Lepidoptera: Gelechiidae) in Ethiopia. Egypt. J. Biol. Pest Control. 2020; 30(1): 1–11. Publisher Full Text\n\nBamisile BS, Dash CK, Akutse KS, et al.: Prospects of endophytic fungal entomopathogens as biocontrol and plant growth promoting agents: An insight on how artificial inoculation methods affect endophytic colonization of host plants. Microbiol. Res. 2018; 217: 34–50. PubMed Abstract \| Publisher Full Text\n\nBehie SW, Jones SJ, Bidochka MJ: Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria. Fungal Ecol. 2015; 13: 112–119. Publisher Full Text\n\nBrownbridge M, Reay SD, Nelson TL, et al.: Persistence of Beauveria bassiana (Ascomycota: Hypocreales) as an endophyte following inoculation of radiata pine seed and seedlings. Biol. Control. 2012; 61(3): 194–200. Publisher Full Text\n\nBruck DJ: Ecology of Metarhizium anisopliae in soilless potting media and the rhizosphere: implications for pest management. Biol. Control. 2005; 32(1): 155–163. Publisher Full Text\n\nCampos-Herrera R, Vicente-Díez I, Galeano M, et al.: Intraspecific virulence of entomopathogenic nematodes against the pests (Thysanoptera: Thripidae) and (Lepidoptera: Gelechiidae). J. Nematol. 2021; 53(1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesneux N, Wajnberg E, Wyckhuys KA, et al.: Biological invasion of European tomato crops by Tuta absoluta: ecology, geographic expansion and prospects for biological control. J. Pest Sci. 2010; 83:197–215. Publisher Full Text\n\nDotaona R, Wilson BA, Stevens MM, et al.: Screening of tropical isolates of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) for virulence to the sweet potato weevil, Cylas formicarius (Coleoptera: Brentidae). Int. J. Trop. Insect Sci. 2015; 35(4): 153–163. Publisher Full Text\n\nEl Kichaoui AY, El-Shafai A, Muheisen H, et al.: Safe approach to the Biological Control of the Tomato Leafminer Tuta absoluta by entomopathogenic fungi Beauveria bassiana isolates from Gaza Strip. Int. J. Appl. Res. 2016; 2: 351–355.\n\nElena GJ, Beatriz PJ, Alejandro P, et al.: Metarhizium anisopliae (Metschnikoff) Sorokin promotes growth and has endophytic activity in tomato plants. Adv. Biol. Res. 2011; 5(1): 22–27.\n\nErdinc C, Ekincialp A, Gundogdu M, et al.: Bioactive components and antioxidant capacities of different miniature tomato cultivars grown by altered fertilizer applications. J. Food Meas. Charact. 2018; 12(3): 1519–1529. Publisher Full Text\n\nGashawbeza A, Abiy F: Occurance and Distribution of a New Species of Tomato Fruit worm, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) in Central Rift Valley of Ethiopia. Proceedings of the 4th Binneial Conference of Ethiopian Horticultural Science Society. 2013, April; pp. 12–13.\n\nGebremariam A, Chekol Y, Assefa F: Phenotypic, molecular, and virulence characterization of entomopathogenic fungi, Beauveria bassiana (Balsam) Vuillemin, and Metarhizium anisopliae (Metschn.) Sorokin from soil samples of Ethiopia for the development of mycoinsecticide. Heliyon. 2021; 7(5): e07091. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeremew D: Arcsine transformed data, evaluation of inoculation techniques of endophytic entomopathogenic fungi, lt50%. Dataset. figshare. 2024. Publisher Full Text\n\nGomez KA, Gomez AA: Statistical procedures for agricultural research. John Wiley & Sons.1984.\n\nGonzalez F, Tkaczuk C, Dinu MM, et al.: New opportunities for the integration of microorganisms into biological pest control systems in greenhouse crops. J. Pest. Sci. 2021; 89(2): 295–311. Publisher Full Text\n\nGreenfield M, Gómez-Jiménez MI, Ortiz V, et al.: Beauveria bassiana and Metarhizium anisopliae endophytically colonize cassava roots following soil drench inoculation. Biol. Control. 2016; 95: 40–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGurulingappa P, Sword GA, Murdoch G, et al.: Colonization of crop plants by fungal entomopathogens and their effects on two insect pests when in planta. Biol. Control. 2010; 55(1): 34–41. Publisher Full Text\n\nHassan FR, Abdullah SK, Assaf LH: Molecular identification and biomass production of an endophytic Beauveria bassiana isolated from cucumber leaves in Iraq. J. Dhaka Univ. 2019; 22(s2): 38–47. Publisher Full Text\n\nKogel KH, Franken P, Hückelhoven R: Endophyte or parasite–what decides? Curr. Opin. Plant Biol. 2006; 9(4): 358–363. Publisher Full Text\n\nLeger RJS: Studies on adaptations of Metarhizium anisopliae to life in the soil. J. Invertebr. Pathol. 2008; 98(3): 271–276. PubMed Abstract \| Publisher Full Text\n\nMansour R, Brévault T, Chailleux A, et al.: Occurrence, biology, natural enemies and management of Tuta absoluta in Africa. Entomol. Gen. 2018; 38(2).\n\nMwamburi LA: Endophytic fungi, Beauveria bassiana and Metarhizium anisopliae, confer control of the fall armyworm, Spodoptera frugiperda (JE Smith)(Lepidoptera: Noctuidae), in two tomato varieties. Egypt. J. Biol. Pest Control. 2021; 31(1): 1–6. Publisher Full Text\n\nOmukoko CA: Biocontrol mechanisms of endophytic Beauveria bassiana in three tomato (Lycopersum esculentum) varieties.2018.\n\nOzdemir IO, Tuncer C, Ozer G: Molecular characterisation and efficacy of entomopathogenic fungi against the Green shield bug Palomena prasina (L.) (Hemiptera: Pentatomidae) under laboratory conditions. Biocontrol. Sci. Tech. 2021; 31(12): 1298–1313. Publisher Full Text\n\nPosada F, Aime MC, Peterson SW, et al.: Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycol. Res. 2007; 111(6): 748–757.\n\nPosada F, Vega FE: Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycoscience. 2006; 47(5): 284–289. Publisher Full Text\n\nResquín-Romero G, Garrido-Jurado I, Delso C, et al.: Transient endophytic colonizations of plants improve the outcome of foliar applications of mycoinsecticides against chewing insects. J. Invertebr. Pathol. 2016; 136: 23–31. PubMed Abstract \| Publisher Full Text\n\nRusso ML, Pelizza SA, Vianna MF, et al.: Effect of endophytic entomopathogenic fungi on soybean Glycine max (L.) Merr. growth and yield. Journal of King Saud University-Science. 2019; 31(4): 728–736. Publisher Full Text\n\nSánchez-Rodríguez AR, Del Campillo MC, Quesada-Moraga E: Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates. Sci. Hortic. 2015; 197: 193–202. Publisher Full Text\n\nSabbour MM: Biocontrol of the tomato pinworm Tuta absoluta (Meyrick)(Lepidoptera: Gelechiidae) in Egypt. Middle East J. Agric. Res. 2014; 3(3): 499–503.\n\nSabbour MM, Singer SM: Evaluations of two isolated Paecilomyces against Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory and field conditions. IJSR. 2014; 3(9):1136–1139.\n\nSaragih M: Endophytic colonization and plant growth promoting effect by entomopathogenic fungus, Beauveria bassiana to Red Chili (Capsicum annuum L.) with different inoculation methods. IOP Conference Series: Earth and Environmental Science. Vol. 305(1): IOP Publishing; 2019, July; p. 012070.\n\nShiberu T, Getu E: Biology of Tuta absoluta (Meyrick)(Lepidoptera: Gelechiidae) under different temperature and relative humidity. J. Hortic. For. 2017; 9(8): 66–73. Publisher Full Text\n\nShiberu T, Getu E: Determination of the economic threshold level of tomato leaf miner, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) on tomato plant under glasshouse conditions. J. Hortic. For. 2018; 10(2): 9–16. Publisher Full Text\n\nShikano I, Rosa C, Tan CW, et al.: Tritrophic interactions: microbe-mediated plant effects on insect herbivores. Annu. Rev. Phytopathol. 2017; 55: 313–331. PubMed Abstract \| Publisher Full Text\n\nSilva ACL, Silva GA, Abib PHN, et al.: Endophytic colonization of tomato plants by the entomopathogenic fungus Beauveria bassiana for controlling the South American tomato pinworm, Tuta absoluta. CABI Agric. Biosci. 2020; 1(1): 1–9.\n\nSinno M, Ranesi M, Di Lelio I, et al.: Selection of endophytic Beauveria bassiana as a dual biocontrol agent of tomato pathogens and pests. Pathogens. 2021; 10(10): 1242. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSousa ASD, Borges SV, Magalhães NF, et al.: Spray-dried tomato powder: reconstitution properties and colour. Braz. Arch. Biol. Technol. 2008; 51(4): 607–614. Publisher Full Text\n\nTefera T, Vidal S: Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus Beauveria bassiana. BioControl. 2009; 54(5): 663–669. Publisher Full Text\n\nVega FE: The use of fungal entomopathogens as endophytes in biological control: a review. Mycologia. 2018; 110(1): 4–30.\n\nVega FE, Posada F, Aime MC, et al.: Entomopathogenic fungal endophytes. Biol. Control. 2008a; 46(1): 72–82. Publisher Full Text\n\nVega FE, Posada F, Aime MC, et al.: Fungal endophytes in green coffee seeds.2008b.\n\nWagner BL, Lewis LC: Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana. Appl. Environ. Microbiol. 2000; 66(8): 3468–3473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmermann G: Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci. Tech. 2007; 17(6): 553–596. Publisher Full Text" }	[ { "id": "304609", "date": "29 Jul 2024", "name": "Yordanys Ramos", "expertise": [ "Reviewer Expertise Biological Control", "Entomology", "Microbiology", "Integrated Pest Management", "Molecular Biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe submitted manuscript describes how the entomopathogenic fungi Beauveria bassiana and Metarhizium robertsii are capable of establishing themselves as endophytes in tomato plants through different artificial inoculation methods while also causing virulence to Tuta absoluta larvae. The results contribute to the integrated management of this pest worldwide. However, there are some aspects that need improvement. Title The authors should consider restructuring the title to make it more informative. An example: Endophytic colonization and establishment of Beauveria bassiana and Metarhizium robertsii against Tuta absoluta (Lepidoptera: Gelechiidae) in tomato plants. Abstract In the results section of the abstract, it is preferable to refer to the isolates as B. bassiana APPRC-27 and M. robertsii K-61. B. bassiana and M. robertsii should be italicized. Correct this throughout the entire document. Additionally, this should be applied to all Latin names used. Keywords In the keywords, avoid repeating words that were previously used in the title of the article, such as colonization, entomopathogenic fungi, and Tuta absoluta. Introduction The statement in the introduction that says, \"Currently, EPF is more promising than chemical pesticides for the control of pests in Ethiopia due to pesticide resistance,\" needs a citation to support it. Methods Conidia were obtained and incubated for 14 days at 27 ± 1°C under dark conditions. At what relative humidity percentage were they conditioned? Were conidia germination tests conducted? Was there any criterion for conidia viability to use them in the bioassays? The authors mention that: \"Tomato leaves surface disinfection was done by washing with 1% sodium hypochlorite for 2 min, 2 min in ethanol (70%), and rinsing 3 times with sterile distilled water.\" However, how did they verify that the sterilization was successful? Typically, this is done by inoculating part of the content of the final rinse water onto PDA or SDA culture media. Please specify the dimensions of the plastic plates used to place the tomato leaves inoculated with the entomopathogenic fungi. You said: \"A control treatment was sprayed with Tween 80 (0.001%) and incubated under the same conditions.\" It should be \"treatment was sprayed with sterile distilled water + Tween 80 (0.001%).\" The authors mention that dead larvae were surface sterilized, but it does not specify how. Was it the same sterilization method as for the tomato leaves mentioned earlier, or was a different method used? Please clarify to avoid ambiguities. The text should provide more information: \"Plates with larval cadavers were moistened daily with distilled sterile water to enhance mycosis development over the cadavers and incubated at room temperature”. Were they incubated in total darkness? Please add the magnification of the stereoscope used. Specify the type of antibiotic and its concentration used in the culture medium when planting the leaves to detect the success of the endophytic establishment of the entomopathogenic fungi. Results The authors mention that \"There is a highly significant difference among treatments in mycosis percentage on dead cadavers.\" Statistical data confirming this statement should be provided. This should be included whenever a comparison is being made. In Figure 2, the standard errors should be included in the lines for each treatment. Conclusions In the conclusions, the authors should avoid repeating the results obtained and be more concise.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "304611", "date": "01 Aug 2024", "name": "Julner Pachoute", "expertise": [ "Reviewer Expertise I have a Ph. D in plant production. I did my thesis on biological control", "entomopathogenic fungi", "endophytic colonization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 part \"EPF from adverse conditions, the incorporation of EPF in plants as endophytes would appear to be a highly interesting and promising approach (Vega, 2018)\"\nThis sentence is not clear, when first mention EPF, it would be better to write it in extension.\nIn the part : Knowing the endophytic association and establishment of EPF in tomato plants helps to modify the inoculation techniques for plant parts based on the behavior of targeted pests, particularly T. absoluta, which lives in the mesophyll tissue of the tomato plant.\nCan yo add some researches about EPF on tomato, especially about B. bassiana and M. robertsi?\nDid you do a literature review about the biological control of T. absoluta? Can you add in the text?\n\nIn the part: 10 km to the west, at a geographical coordinate of 8°59’ N latitude and 37.85°E longitude, with an altitude of 2100 meters above sea l Revise the coordinates?\nIn the part: Conidia were obtained from pure cultures grown on the 65g of Sabouraud Dextrose Agar (SDA) Microxpress® REF: 2\nConidia of what? Please cite the fungus.\n\nIn the part: The mass rearing of T. absoluta was established in 2021 from larvae collected from tomato plants without any history of pesticides in fields surrounding Ambo areas. The Roman VF variety tomato plant was grown in the glasshouse to maintain the life cycle of T\nPlease describe the collet of the larvae T. absoluta\n\nIn the part: High-virulent B. bassiana and M. robertsii isolates that were prescree\nCan you talk more about the origin of the isolates\n\nIn the section: Currently, EPF is more promising than chemical pesticides for the control of pests in Ethiopia due to pesticide resistance. Are you sure? Do you have more augment ou reference to support this thesis.\n\nIn the section: The result is consistent with the fact that Metarhizium species prefer root tissues to leaves as endophytic colonization after artificial inoculation, while B. bassiana prefers aerial plant parts (Behie et al., 2015). M. robertsii has a well-established relationship with the rhizosphere (Leger, 2008; Bruck, 2005). However, successful endophytic colonization of B. bassiana occurred in tomato root tissue at 37.75% at 7 dpi by the root dipping method (Mwamburi, 2021).\nCan you explain this preference?\nIn the section: all inoculation techniques, this study revealed that B. bassiana and M. robertsii can migrate endophytically from the inoculated sites and colonize other plant tissues in the tomato plant. This supports the systematic translocation and colonization of fungal inoculum's' in plant tissues from sites inoculated to other plant tissues (Tefera and Vidal, 2009; Sánchez-Rodríguez et al., 2015; Omukoko, 2018; Sinno et al., 2021). Elena et al.(2011) also reported that M. robertsii was capable of moving within the plant and B. bassiana was able to colonize corn and maize within plant tissues by stem injection technique.\nIs it a good idea to discuss the results comparing different species?\nIn the section: the potential to colonize and migrate in tomato plant tissues. However, endophytic colonization reduced over time at 28 days post-inoculation for all isolates by leaf spray and root dipping techniques while increasing by seedling inoculation techniques.\nCan you explain why?\nProvide photos with scale bar.\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": "304615", "date": "05 Aug 2024", "name": "Eustachio Tarasco", "expertise": [ "Reviewer Expertise microbiological control", "insect pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBibliography needs to be increased as well as systematics (each species when reported for the first time in the text should be written in full with Authority and systematics) and details on the origin and characterization of the entomopathogenic fungi used in the experiments. The methodology can be better explained with a more detailed experimental design. Moreover too short conclusions. The last 2 chapters could be better arranged\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "304612", "date": "26 Aug 2024", "name": "Preety Tomar", "expertise": [ "Reviewer Expertise Entomopathogens", "Biocontrol", "Microbes", "Crop protection" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research article entitled \"Evaluation of endophytic colonization and establishment of entomopathogenic fungi against Tuta absoluta (Lepidoptera: Gelechiidae) in tomato plants\" is a very good work carried by Greremew et al. The authors have selected an important area of investigation which is the management of leaf miner larvae using entomopathogens. The results are supplemented by tables and figures including nice photographs. The only thing that i want to suggest is about the writing of scientific names. They should be italic and should be written as whole only once after that they should be written as short form. There are some typographical errors though they do not belittle the importance and genuineness of the work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-800
https://f1000research.com/articles/13-799/v1	16 Jul 24	{ "type": "Systematic Review", "title": "Beyond stroke therapy, neuroaid (a chinese herbal) has an effect on cognition and neurogenesis, a bibliometric study", "authors": [ "ARMAN YURISALDI SALEH", "Riezky Valentina", "Tirta Darmawan Susanto", "Dwi Arwandi Yogi Saputra", "Riezky Valentina", "Tirta Darmawan Susanto", "Dwi Arwandi Yogi Saputra" ], "abstract": "Introduction NeuroAiD, also known as MLC601 or MLC901, is a Chinese herbal combination used worldwide for stroke treatment. It contains herbal components and five hewan components. MLC601 contains herbal components and hewan components, while MLC901 has a similar herbal composition. NeuroAiD is used to support neurologic recovery after stroke and to aid cognitive function in Alzheimer’s disease. Studies show that NeuroAiD has potential in treating Alzheimer’s disease and is beneficial in both local and global stroke models and in the Kortikal culture. However, there is limited bibliometric research on NeuroAiD, which is a method of collecting data from published articles to analyze developments and trends in the field of research. This research contributes significantly to the literature and helps develop more effective stroke treatment strategies.\n\nMethods In this work, a literature review methodology is employed to gather data from the Scopus database using the keywords neuroaid. Data were analyzed using Biblioshiny and VOSviewer software to produce visualizations and bibliometric maps. We conducted quantitative and qualitative analysis\n\nResults The research trend found are documents by year, most relevant sources, factorial map of the most cited documents, factorial map of The documents with the highest contributes, documents by author, documents by country or territory, documents by subject area, documents by affiliation, network visualization, overlay visualization of scopus database using vosviewer, density visualization, thematic map, thematic evolution, topic dendogram, and world cloud.\n\nConclusions The study investigates the potential of Neuroaid, a neuroprotective drug, for stroke prevention and cognitive function enhancement. It uses terms like “cognition” and “neurogenesis” to highlight its potential. While the study’s focus may be limited, it provides valuable insights into research direction and potential areas of neuroaid for stroke treatment.", "keywords": [ "neuroaid", "cognitive enhancement", "stroke", "neuroprotective", "bibliometric" ], "content": "Introduction\n\nNeuroAiD, also known as MLC601 or MLC901, is a combination of traditional Chinese herbs that has been used widely throughout the world for stroke therapy.1,2 NeuroAiD contains nine herbal plants that help in blood circulation.1\n\nNeuroAiD (MLC601 and MLC901) is a drug derived from traditional Chinese medicine. Both formulations have herbal components, but there are differences in their composition.\n\nMLC601 consists of nine herbal components and five animal components. The herbal components include: Radix astragali, Radix salviae miltiorrhizae, Radix paeoniae rubra, Rhizoma chuanxiong, Radix angelicae sinensis, Carthamus tinctorius, Prunus persica, Radix polygalae, and Rhizoma acori tatarinowii.\n\nAnimal components in MLC601 include Hirudo, Eupolyphaga seu steleophaga, Calculus bovisartifactus, Buthus martensii, and Cornu saigae tataricae.3\n\nMLC901 is a simplified version of MLC601 and contains only the same nine herbal components as MLC601.1,4\n\nBoth forms have demonstrated neuroprotective and neurodegenerative effects in cerebral ischemia models.3,5 However, MLC901 has several advantages that may improve patient compliance.6 Although MLC601 and MLC901 have similar safety and efficacy profiles, MLC901 does not contain animal components, which makes it more acceptable to patients who may have objections to the use of animal products.6 This drug has been used extensively to facilitate neurological recovery after stroke, especially in the non-acute phase. Additionally, NeuroAiD has also been used to support cognitive function in Alzheimer’s disease.\n\nClinical and preclinical studies suggest that NeuroAiD has a role in Alzheimer’s disease. Additionally, NeuroAiD has demonstrated neuroprotective and neuroregenerative properties in animal models of focal and global ischemia as well as in cortical cell cultures. These properties are critical in developing treatment strategies to reduce long-term disability from stroke, heart attack, and other brain injuries.\n\nAlthough NeuroAiD has been widely researched, no bibliometric studies have been conducted using documents from www.scopus.com. Bibliometric studies are a research method that uses data from scientific publications to analyze developments and trends in a particular research field. In this context, bibliometric studies can provide valuable insight into NeuroAiD research and how its use has evolved over time.\n\nThis research is especially important as there is increasing research on NeuroAiD and an increasing number of neurology centers using NeuroAiD for stroke therapy. Apart from that, this research also aims to look at the possibility of using NeuroAiD as a standard stroke therapy in the future. Thus, this study hopes to make a significant contribution to the existing literature and assist in the development of more effective stroke treatment strategies.\n\n\nMethods\n\nBibliometric research is a research method that uses scientific publication data to describe and analyze the development of a field of science. This research aims to identify and map trends, patterns, and relationships between scientific documents related to certain topics. In this research, the topic chosen was neuroaid. This research uses data from the website www.scopus.com, which is one of the largest and most trusted databases for scientific publications. This research was conducted on early May 2024.\n\nTo carry out bibliometric research, the steps to follow are as follows:\n\n1. Determine search keywords. In this research, the keywords used are neuroaid. These keywords are entered into the search column on the www.scopus.com site by selecting the topic field (title, abstract, keywords).\n\n2. Filter search results. In this study, Were not filtered.\n\n3. Download search results data. In this research, search result data is downloaded in three different formats, namely:\n\n• CSV (comma-separated value), which contains basic information about the document, such as title, author, affiliation, year, source, abstract, and keywords.\n\n• RIS (research information system), which contains detailed information about a document, including the references cited by the document.\n\nData for this study were collected by one reviewer. Reviewers work independently in collecting data from each report. To ensure data accuracy and clutter. In addition, we use automated tools, namely the VosViewer and Biblioshiny applications, to assist in the data collection and analysis process.\"\n\nin this study we found are documents by year has been an increase in the number of documents; until 2023, there were 9 documents, most relevant sources is journal of cerebrovascular diseases, documents by author with the most authors with 30 documents are Venketasubramanian, N., documents by country or territory is singapore is the country with the largest number of document producers 37 documents, documents by subject area, network visualization, overlay visualization of scopus database using vosviewer, density visualization, thematic map based on the title shows that the niche theme is the keyword mitochondria signaling pathway, effects neuroprotective effect, dan oxidative stres toxicity, thematic evolution, cluster analysis, qualitative analysis, and word cloud\n\nWe used the following terms to do a search on the Scopus website, taking into consideration that this website contains research that is considered to be valid: TITLE – ABS – KEY (neuroaid) are the titles of the products that are under consideration. Ninety-two documents were received by us. We then save the document from Scopus in the form of a file with the extension.csv file following this step.\n\nBoth the Biblioshiny and Vosviewer software packages were utilised in the analysis process.\n\nDocuments by year\n\nBased on Figure 1, it appears that there has been an increase in the number of documents, until in 2023 there were 9 documents. The oldest document in 2008 was entitled Neuroaid in stroke recovery., written by Siow, C.H.C.,7 Next is the article entitled Danqi Piantan Jiaonang does not modify hemostasis, hematology, and biochemistry in normal subjects and stroke patients written by Gan, R. et al.8 Meanwhile, the latest document in 2024 is entitled Facilitation of neurological recovery in a complete spinal cord injury with NeuroAiD: case report written by Zainudin, M.F., Abu Hassan, S.A., Khin, N.Y.9\n\nMost relevant sources\n\nBased on Figure 2, The following are the journals that publish the most important documents: The first is the Journal of Cerebrovascular Diseases. This journal is indexed in Scopus and is published by Karger Publisher. The SJR (SCImago Journal Rank) of this journal for 2023 is around 0.25. In 2023, this journal will be in the Q2 quartile for the cardiology and cardiovascular medicine category. The head office of Karger Publishers is located in Switzerland. The journal accepts manuscripts on topics covering prevention, diagnosis, management, rehabilitation, and related fields of molecular genetics, vascular biology, pathophysiology, epidemiology, and health systems science. The journal welcomes original research, reviews, and commentary articles focused on improving knowledge and information about clinical practice and health policy. The review time for this journal until the first decision is around 16.2 days. The H-index of this journal is 119. And the impact factor of this journal is around 2.96 in 2023.\n\nThe next journal is the International Journal of Stroke. This journal is indexed in Scopus and published by SAGE Publications Ltd. The SJR (SCImago Journal Rank) for 2023 is around 1,952. As of 2023, the journal is in the Q1 quartile for the categories “Neurology” and “Neurology (clinical). The head office of SAGE Publications is located in the United Kingdom. The journal accepts manuscripts on topics covering clinical aspects of stroke from around the world with basic scientific contributions in the field of clinical interest. This journal accepts reviews of current topics, leading opinions, research, panoramas, clinical trial protocols, and guidelines. The time for this journal’s review to the first decision is around 15.0 days from this journal, which is around 6,948 in 2023.\n\nThe next journal is the Stroke Journal. This journal has been indexed in Scopus since 1970. This journal is published by the American Heart Association. The SJR (SCImago Journal Rank) for 2023 is around 2,746. In 2023, this journal will be in the Q1 quartile for the categories Advanced and Specialized Nursing, Cardiology and Cardiovascular Medicine, Medicine (miscellaneous), Neurology (clinical), and Neuroscience (miscellaneous). The headquarters of the American Heart Association are located in the United States. The journal accepts manuscripts on topics covering all aspects of cerebral circulation and its diseases from many disciplines, including neurology, internal medicine, radiology, nuclear medicine, neuropathology, neurosurgery, epidemiology, vascular surgery, rehabilitation, anesthesiology, critical care, vascular physiology, neuropsychology, speech pathology, and neuro-ophthalmology. The H-index of this journal is 357. And the impact factor of this journal is around 10.17 in 2023.\n\nFactorial map of the most cited documents\n\nBased on Figure 3, the document with the most citations based on factorial analysis is The NeuroAiD II (MLC901) in Vascular Cognitive Impairment Study (NEURITES) by Chen CLH, Ikram K, Anqi Q, Yin WT, Chen A, and Venketasubramanian N, which was published in Cerebrovasc Dis in 2013.10,11\n\nThis article discusses the NEURITES study, a 24-week, double-blind, randomized, placebo-controlled phase II study of NeuroAiD II in patients with vascular cognitive impairment (VCIND). The aim of this study was to investigate the effects and tolerability of NeuroAiD II in patients with VCIND.11\n\nNeuroAiD is a traditional Chinese medicine that has been shown to induce neuroplasticity, stimulate cell proliferation, and stimulate the development of dense axial and dendritic tissue in animal stroke models. NeuroAiD may be able to improve cerebral blood flow and functional recovery after stroke in patients.11\n\nThe main outcome of this study was executive function as measured by the verbal fluency test. Secondary outcomes include cognitive assessments such as ADAS-Cog, MoCA, MMSE, and Cognitive Battery; daily activities as measured by the Alzhei-Mer’s Disease Cooperative Study Activities of Daily Living (ADCS-ADL) scale for mild cognitive impairment; behavior as measured by the Neuropsychiatric Inventory; and depression as measured by the Geriatric Depression Scale and Beck Depression Scale.11\n\nThe NEURITES study has the potential to set new standards for the systematic evaluation of traditional Asian medicines for integration into standard medical practice and establish new therapeutic approaches to improve cognition after stroke.11\n\nFactorial map of the documents with the highest contributes\n\nIn Figure 4, the document with the highest contributor is the document entitled The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX, BCL-2, BAD, PGC-1α, and TFAM Genes in the Hippocampus of Male Wistar Rats by Molaei P, Vaseghi S, Entezari M, Hashemi M, and Nasehi M, published in Neurochem Res, volume 46, pages 2154-2166, on May 24, 2021.12\n\nDocuments by author\n\nBased on Figure 5 the most authors with 30 documents are Venketasubramanian, N., with the titles of several articles, namely: T Association between Baseline NIHSS Limb Motor Score and Functional Recovery after Stroke: Analysis Based on a Multicountry Dataset,13 Ischemic Stroke and Savings in Time to Achieve Functional Recovery: Experience from NeuroAiD,14 Alzheimer’s Disease THErapy With NEuroaid (ATHENE): A Randomized Double-Blind Delayed-Start Trial,15 NEURoaid II (MLC901) in cognitively Impaired not demenTEd patientS (NEURITES): A pilot double blind, placebo-controlled randomized trial,16 and NeuroAid II (MLC901) in Haemorrhagic Stroke.17\n\nThe next most author with 20 documents is Chen, C.L.H., with the titles of several articles, namely: Association between Baseline NIHSS Limb Motor Score and Functional Recovery after Stroke: Analysis Based on a Multicountry Dataset,13 Ischemic Stroke and Savings in Time to Achieve Functional Recovery: Experience from NeuroAiD,14 Alzheimer’s Disease THErapy With NEuroaid (ATHENE): A Randomized Double-Blind Delayed-Start Trial,15 NEURoaid II (MLC901) in cognitively Impaired not demenTEd patientS (NEURITES): A pilot double blind, placebo-controlled randomized trial,16 and article title Frequency and clinical impact of serious adverse events on post-stroke recovery with NeuroAiD (MLC601) versus Placebo: The CHInese medicine neuroaid efficacy on stroke recovery study.18\n\nDocuments by country or territory\n\nBased on Figure 6, Singapore is the country with the largest number of document producers 37 documents. Followed by France with 17 documents, Iran with 16 documents, Malaysia with 13 documents, and the Philippines with 12 documents.\n\nDocuments by subject area\n\nDocuments by affiliation\n\nBased on Figure 8, in first place, the producer of the most documents is affiliated with the National University of Singapore with 30 documents, next in second place is affiliated with Raffkes Hospital, Singapore with 17 documents and next is the National Neuroscience Institute of Singapore with 13 documents.\n\nNetwork visualization\n\nBased on Figure 9, it can be seen that the areas studied are still not related to other areas that are divided into edges. That field is: stroke rehabilitation, time to treatment, time factor, procedures, severity of illness index, recovery, in vitro study, mice inbred c57bl, animal cell, mice, disease models animal, pathology, disease model, rats, apoptosis, rats wistar, wistar rat, gene expression, traumatic brain injury, safflower, cognitive defect, nuclear magnecti resonance imaging, neuroaid ii, executive function, nootropic agent, polygalacea, pilot study, drug tolerability, abdominal discomfort, safety, side effect, antihypertensive agent, traditional chinese medicine, brain injuries, hippocampus, cohort analysis, systematic review, brain infarction, brain, and human cell.\n\nOverlay visualization of scopus, database using vosviewer\n\nBased on Figure 10, in the overlay visualization, it appears that the keywords that are being researched a lot approaching 2019 are the parts colored yellow, namely: mic901, animal, pscychology, hippocampus, pilot study, cognition, wistar rat, disability, and neuroaid ii.\n\nDensity visualization\n\nBased on Figure 11, in the visual circulation density, it appears that the part that is already saturated with research is yellow, while the part that is not yet saturated is slightly yellow and dominantly green, namely keywords: stroke rehabilitation, time to treatment, time factor, time factors, procedures, recovery, drug effects, mice inbredc57bl, in vitro study, animal cell, mouse, mice, pathology, cell proliferation, rats, apoptosis, rat, rats wistar, hippocampus, wistar rat, gene expression, neurogenesis, safflower, cognitive defect, nuclear magnetic resonance ima, patient compliance, neuroaid ii, executive function, nootropic agent, pilot study, polygalaceae, naursea, safety, drug tolerability, abdominal discomfort, vomiting, side effect, antihypertensive agent, traditional chinese medicine, stroke recovery, severity of illness index, and cohort analysis.\n\nThematic map\n\nBased on Figure 12, in the niche section there are the keywords cognition, neurogenesis then the keywords effect, treatment, rats then the keywords registry, safe.\n\nThematic evolution\n\nBased on Figure 13, there was an evolution of changes in themes in research in 2008-2017 with the keywords mlc, neuroaid, cognitive, patients, and positive. The theme then changed in 2018-2024 to neuroaid, mlc, recovery, pilot and brain.\n\nTopic dendogram\n\nBased on Figure 14, there are 2 large clusters based on keywords. There are 2 clusters of blue and red.\n\nWord cloud\n\nBased on Figure 15, word cloud shows that the dominance in the document is neuroaid, mlc, stroke, recovery, and study.\n\n\nDiscussion\n\nThe niche theme in this study represents a more specific and perhaps less explored area of research in the current literature. For example, keywords such as “cognition” and “neurogenesis” may indicate that there is ongoing research into how neuroaid may affect cognitive function and the growth of new nerve cells. Meanwhile, keywords like “treatment,” “rats,” and “registry safe” may indicate that there is ongoing research into Neuroaid’s use in treatment, testing in mice, and its safety.\n\nThe motor theme includes keywords such as “brain,” “injury,” and “cognitive,” which may indicate that there is a lot of research that has been done on how Neuroaid can aid in recovery from brain injury and improvement of cognitive function. Other keywords such as “MLC,” “study,” “efficacy,” “Chinese,” “medicine,” “traditional,” “neuroaid,” “stroke,” and “recovery” indicate that there is extensive research on the efficacy of Neuroaid as a Traditional Chinese medicine in stroke recovery.\n\nThemes that emerged or decreased were “MLC,” “study,” and “efficacy.” This suggests that research focus may be shifting in this direction or may indicate that research in this area may be declining. For example, if there is a decline in research on the efficacy of Neuroaid, this may indicate that researchers may be switching to another drug or therapy.\n\nHowever, it is important to note that the results from this study may change if different keywords are used. This means that the interpretation of this study should be done with caution, considering that the results may be different if different keywords are used. For example, if the keyword “rehabilitation” is used instead of “recovery,” the results may be very different.\n\nAdditionally, this bibliometric study is new and uses document data from www.scopus.com. Therefore, the results may not reflect the entire literature available outside of this data source. For example, if there is research published in a journal that is not indexed in Scopus, it will not be included in this study. Nonetheless, this study provides valuable insight into current research trends and potential future research directions in the field of Neuroaid.\n\nNeuroids, which were originally used to treat ischemic stroke, have now been used to treat hemorrhagic stroke, intelligent spinal injury, vascular dementia, Parkinson’s, traumatic brain injury, cognitive impairment, hypoxic ischemic brain injury, epilepsy, cardioprotective, and Alzheimer’s. With this bibliometric study, it is hoped that it can increase clinical insight so that neuroids can be included in recommendations and various expert consensuses in fields other than stroke.\n\n\nConclusion\n\nThe study focuses on specific research areas and may have limited research on the topic. Key terms include “cognition” and “neurogenesis,” which indicate that neuroaid can influence cognitive function and new behaviors. Key terms include “treatment,” “rats,” and “registry safe,” which indicate that Neuroaid can help in stroke prevention and cognitive function enhancement. Key terms include “MLC,” “study,” and “efficacy,” which indicate that the focus of the study may be limited or irrelevant. However, the study’s findings may differ if different research bodies are used. Interpretation should be done with caution, considering the differences between the two groups. The study also uses bibliometric data from Scopus.com, which may not cover all available literature. Despite this, the study provides valuable insights into the research direction and potential areas of research in neuroscience. Neuroids are used for stroke prevention, hemorrhagic stroke, spinal cord injuries, vascular dementia, Parkinson’s, traumativ brain injury, cognitive impairment, hypoxic ischemic brain injury, epilepsi, cardioprotective, and Alzheimer’s. The bibliometric study can help create a consensus on neuroids for stroke treatment.\n\nAYS conducts research, gathers data, performs statistical analysis, and produces discussions and conclusions, RV, TDS and DAYS editing.", "appendix": "Data availability statement\n\nFigshare: Beyond stroke therapy, neuroaid (a chinese herbal) has an effect on cognition and neurogenesis, a bibliometric study, https://doi.org/10.6084/m9.figshare.25943860.v1. 88\n\nFigshare: PRISMA-ScR checklist: Beyond stroke therapy, neuroaid (a chinese herbal) has an effect on cognition and neurogenesis, a bibliometric study: https://doi.org/10.6084/m9.figshare.25943869.v1. 89\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nKumar R, Abu Bakar A, Thanabalan J, et al.: Safety and Use of MLC601/MLC901 (NeuroAiDTM) in Primary Intracerebral Hemorrhage: A Cohort Study from the NeuroAiD Safe Treatment Registry. Brain Sci. 2020; 10(8). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Tao W, Zhang J, et al.: Neuroaid for improving recovery after ischemic stroke. Cochrane Database Syst. Rev. 2017 Aug 26; 2017(8): CD009757. eCollection 2017 Aug. Publisher Full Text\n\nPilipenko P, Ivanova AA, Kotsiubinskaya YV, et al.: Randomised, double-blind, placebo-controlled study investigating Safety and efficAcy of MLC901 in post-traUmatic bRAin Injury: the SAMURAI study protocol. BMJ Open. 2022; 12(4): e059167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan C-Hoon N, Choy D, Venketasubramanian N: NeuroAid II (MLC901) in Haemorrhagic Stroke. Case Rep. Neurol. 2020; 12(Suppl. 1): 212–217. Publisher Full Text\n\nRanuh I, Sari GM, Utomo B, et al.: Systematic Review and Meta-Analysis of the Efficacy of MLC901 (NeuroAiD II (TM)) for Acute Ischemic Brain Injury in Animal Models. J. Evid.-Based Integr. Med. 2021; 26: 2515690X2110392. Publisher Full Text\n\nKumar R, Abu Bakar A, Thanabalan J, et al.: Safety and Use of MLC601/MLC901 (NeuroAiD (TM)) in Primary Intracerebral Hemorrhage: A Cohort Study from the NeuroAiD Safe Treatment Registry. Brain Sci. 2020; 10(8). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiow CHC: Neuroaid in Stroke Recovery. Eur. Neurol. 2008; 60(5): 264–266. Publisher Full Text\n\nGan R, Lambert C, Lianting J, et al.: Danqi Piantan Jiaonang does not modify hemostasis, hematology, and biochemistry in normal subjects and stroke patients. Cerebrovasc. Dis (Basel, Switzerland). 2008; 25(5): 450–456. PubMed Abstract \| Publisher Full Text\n\nZainudin MF, Abu Hassan SA, Khin NY: Facilitation of neurological recovery in a complete spinal cord injury with NeuroAiD: case report. Spinal Cord Ser. Cases. 2024; 10(1): 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CL, Ikram K, Anqi Q, et al.: The NeuroAiD II (MLC901) in vascular cognitive impairment study (NEURITES). Cerebrovasc. Dis (Basel, Switzerland). 2013; 35 Suppl 1: 23–29. PubMed Abstract \| Publisher Full Text\n\nChen C, Ikram K, Qiu A, et al.: The NeuroAiD II (MLC901) in vascular cognitive impairment study (NEURITES) for the NEURITE investigators. Cerebrovasc. Dis. 2013; 35: 23–29. PubMed Abstract \| Publisher Full Text\n\nMolaei P, Vaseghi S, Entezari M, et al.: The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX, BCL-2, BAD, PGC-1α and TFAM Genes in the Hippocampus of Male Wistar Rats. Neurochem. Res. 2021; 46(8): 2154–2166. PubMed Abstract \| Publisher Full Text\n\nChen CLH, Pokharkar Y, Venketasubramanian N: Association between Baseline NIHSS Limb Motor Score and Functional Recovery after Stroke: Analysis Based on a Multicountry Dataset. Cerebrovasc. Dis (Basel, Switzerland). 2023; 52(2): 160–165. PubMed Abstract \| Publisher Full Text\n\nVenketasubramanian N, Pokharkar Y, Chai JH, et al.: Ischemic Stroke and Savings in Time to Achieve Functional Recovery: Experience from NeuroAiD. J. Cardiovasc. Dev. Dis. 2023; 10(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CLH, Lu Q, Moorakonda RB, et al.: Alzheimer’s Disease THErapy With NEuroaid (ATHENE): A Randomized Double-Blind Delayed-Start Trial. J. Am. Med. Dir. Assoc. 2022; 23(3): 379–86.e3. PubMed Abstract \| Publisher Full Text\n\nChen CLH, Nguyen TH, Marasigan S, et al.: NEURoaid II (MLC901) in cognitively Impaired not demenTEd patientS (NEURITES): A pilot double blind, placebo-controlled randomized trial. Alzheimer’s & dementia (New York, N Y). 2021; 7(1): e12161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan CN, Choy D, Venketasubramanian N: NeuroAid II (MLC901) in Haemorrhagic Stroke. Case Rep. Neurol. 2020; 12(Suppl 1): 212–217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenketasubramanian N, Moorakonda RB, Lu Q, et al.: Frequency and Clinical Impact of Serious Adverse Events on Post-Stroke Recovery with NeuroAiD (MLC601) versus Placebo: The CHInese Medicine Neuroaid Efficacy on Stroke Recovery Study. Cerebrovasc. Dis (Basel, Switzerland). 2020; 49(2): 192–199. PubMed Abstract \| Publisher Full Text\n\nNasehi M, Torabinejad S, Hashemi M, et al.: Effect of cholestasis and NeuroAid treatment on the expression of Bax, Bcl-2, Pgc-1α and Tfam genes involved in apoptosis and mitochondrial biogenesis in the striatum of male rats. Metab. Brain Dis. 2020; 35(1): 183–192. PubMed Abstract \| Publisher Full Text\n\nNasehi M, Mohammadi A, Ebrahimi-Ghiri M, et al.: MLC901 during sleep deprivation rescues fear memory disruption in rats. Naunyn Schmiedeberg’s Arch. Pharmacol. 2019; 392(7): 813–821. PubMed Abstract \| Publisher Full Text\n\nKumar R, Htwe O, Baharudin A, et al.: Spinal cord injury - assessing tolerability and use of combined rehabilitation and NeuroAiD (SATURN) study - primary results of an exploratory study. J. Spinal Cord Med. 2023; 46(4): 682–686. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPakdaman H, Amini Harandi A, Gharagozli K, et al.: MLC601 in vascular dementia: an efficacy and safety pilot study. Neuropsychiatr. Dis. Treat. 2017; 13: 2551–2557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumari E, Shukla MK, Pandey OJ, et al.: NeuroAid: Emotion-Based EEG Analysis for Parkinson’s Disease Identification. IEEE Sensors Letters.IEEE Sens. Lett. 2023; 7: 1–4. Publisher Full Text\n\nPonsford J, Velikonja D, Janzen S, et al.: INCOG 2.0 Guidelines for Cognitive Rehabilitation Following Traumatic Brain Injury, Part II: Attention and Information Processing Speed. J. Head Trauma Rehabil. 2023; 38(1): 38–51. PubMed Abstract \| Publisher Full Text\n\nTheadom A, Barker-Collo S, Jones KM, et al.: MLC901 (NeuroAiD II™) for cognition after traumatic brain injury: a pilot randomized clinical trial. Eur. J. Neurol. 2018; 25(8): 1055–e82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuintard H, Lorivel T, Gandin C, et al.: MLC901, a Traditional Chinese Medicine induces neuroprotective and neuroregenerative benefits after traumatic brain injury in rats. Neuroscience. 2014; 277: 72–86. PubMed Abstract \| Publisher Full Text\n\nChio CC, Lin MT, Chang CP, et al.: A positive correlation exists between neurotrauma and TGF-β1-containing microglia in rats. Eur. J. Clin. Investig. 2016; 46(12): 1063–1069. PubMed Abstract \| Publisher Full Text\n\nChua AE, Yacapin VJ, Manalo GL 3rd, et al.: Protocol for Safety and Efficacy of MLC901 (NeuroAiD II) in Patients With Moderate Traumatic Brain Injury: A Randomized Double-Blind Placebo-Controlled Trial (ANDROMEDA). Neurosurgery. 2023; 93(4): 939–951. PubMed Abstract \| Publisher Full Text\n\nRosyidi RM, Priyanto B, Islam AA, et al.: Role of MLC901 in increasing neurogenesis in rats with traumatic brain injury. Ann. Med. Surg. 2012; 2020(60): 36–40.\n\nTsai MC, Chang CP, Peng SW, et al.: Therapeutic efficacy of Neuro AiD™ (MLC 601), a traditional Chinese medicine, in experimental traumatic brain injury. J. Neuroimmune Pharmacol. 2015; 10(1): 45–54. PubMed Abstract \| Publisher Full Text\n\nOgbole G, Efidi R, Odo J, et al.: Brain computed tomography perfusion analysis in HIV-seropositive adults with and without neurocognitive impairment in Nigeria: outcomes and challenges of a pilot study. Pan Afr. Med. J. 2023; 46: 15.\n\nPakdaman H, Amini Harandi A, Abbasi M, et al.: Efficacy and Safety of MLC601 in the Treatment of Mild Cognitive Impairment: A Pilot, Randomized, Double-Blind, Placebo-Controlled Study. Dement. Geriatr. Cogn. Dis. Extra. 2017; 7(1): 136–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPakdaman H, Amini Harandi A, Gharagozli K, et al.: A Long-term Study of NeuroAid (MLC601, MLC901) in Patients with Alzheimer’s Disease; An Extension 8-year Follow-up Study. Curr. Aging Sci. 2023; 16(3): 234–239. PubMed Abstract \| Publisher Full Text\n\nMalboosi N, Nasehi M, Hashemi M, et al.: The neuroprotective effect of NeuroAid on morphine-induced amnesia with respect to the expression of TFAM, PGC-1α, ΔfosB and CART genes in the hippocampus of male Wistar rats. Gene. 2020; 742: 144601. PubMed Abstract \| Publisher Full Text\n\nNasehi M, Zadeh-Tehrani SN, Khakpai F, et al.: A possible neuroprotective property of ethanol and/or NeuroAiD on the modulation of cognitive function. Neurotoxicol. Teratol. 2020; 82: 106927. PubMed Abstract \| Publisher Full Text\n\nPakdaman H, Gharagozli K, Karamiani F, et al.: MLC901 in hypoxic-ischemic brain injury patients: A double-blind, randomized placebo-controlled pilot study. Medicine. 2023; 102(23): e33914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFroestl W, Pfeifer A, Muhs A: Cognitive enhancers (nootropics). Part 3: drugs interacting with targets other than receptors or enzymes. disease-modifying drugs. J. Alzheimers Dis. 2013; 34(1): 1–114. Publisher Full Text\n\nSalim Mahmood A, Ammoo AM, Ali MHM, et al.: Antiepileptic Effect of Neuroaid® on Strychnine-Induced Convulsions in Mice. Pharmaceuticals (Basel, Switzerland). 2022; 15(12). Publisher Full Text\n\nHeshmatzad K, Nasehi M, Vaseghi S: Effects of morphine and NeuroAid on the expression levels of GluN2A and GluN3A in the hippocampus and striatum of rats. Iran. J. Basic Med. Sci. 2021; 24(4): 469–475. PubMed Abstract \| Publisher Full Text\n\nVincent A, Covinhes A, Barrère C, et al.: Acute and long-term cardioprotective effects of the Traditional Chinese Medicine MLC901 against myocardial ischemia-reperfusion injury in mice. Sci. Rep. 2017; 7(1): 14701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPakdaman H, Gharagozli K, Abbasi M, et al.: Efficacy and Safety of MLC601 in Patients with Mild to Moderate Alzheimer Disease: An Extension 4-Year Follow-Up Study. Dement. Geriatr. Cogn. Dis. Extra. 2018; 8(1): 174–179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee WT, Hsian CCL, Lim Y-A: The effects of MLC901 on tau phosphorylation. NeuroReport. 2017; 28(16): 1043–1048. PubMed Abstract \| Publisher Full Text\n\nChen CLH, Sharma PR, Tan BY, et al.: The Alzheimer’s disease THErapy with NEuroaid (ATHENE) study protocol: Assessing the safety and efficacy of Neuroaid II (MLC901) in patients with mild-to-moderate Alzheimer’s disease stable on cholinesterase inhibitors or memantine-A randomized, double-blind, placebo-controlled trial. Alzheimers Dement (New York, N Y). 2019; 5: 38–45. PubMed Abstract \| Publisher Full Text\n\nLim YA, Murray LA, Lai MK, et al.: NeuroAiD® (MLC601) and amyloid precursor protein processing. Cerebrovasc. Dis (Basel, Switzerland). 2013; 35 Suppl 1: 30–37. PubMed Abstract \| Publisher Full Text\n\nChen CL, Young SH, Gan HH, et al.: Chinese medicine neuroaid efficacy on stroke recovery: a double-blind, placebo-controlled, randomized study. Stroke. 2013; 44(8): 2093–2100. PubMed Abstract \| Publisher Full Text\n\nVenketasubramanian N, Young S, Tay SS, et al.: Chinese medicine NeuroAiD efficacy stroke recovery-extension study (CHIMES-E study): an observational multicenter study to investigate the longer-term efficacy of NeuroAiD in stroke recovery. Cerebrovasc. Dis (Basel, Switzerland). 2013; 35 Suppl 1: 18–22. PubMed Abstract \| Publisher Full Text\n\nLee CF, Venketasubramanian N, Wong KS, et al.: Comparison Between the Original and Shortened Versions of the National Institutes of Health Stroke Scale in Ischemic Stroke Patients of Intermediate Severity. Stroke. 2016; 47(1): 236–239. PubMed Abstract \| Publisher Full Text\n\nChen C, Venketasubramanian N, Gan RN, et al.: Danqi Piantang Jiaonang (DJ), a traditional Chinese medicine, in poststroke recovery. Stroke. 2009; 40(3): 859–863. PubMed Abstract \| Publisher Full Text\n\nKong KH, Wee SK, Ng CY, et al.: A double-blind, placebo-controlled, randomized phase II pilot study to investigate the potential efficacy of the traditional chinese medicine Neuroaid (MLC 601) in enhancing recovery after stroke (TIERS). Cerebrovasc. Dis (Basel, Switzerland). 2009; 28(5): 514–521. PubMed Abstract \| Publisher Full Text\n\nAbdul Rashid AM, Md Noh MSF, Yusof Khan AHK, et al.: NeuroAid II (MLC901) and polypharmacy in stroke and the risk of hepatotoxicity: a case report. Egypt. J. Neurol. Psychiat. Neurosurg. 2021; 57(1): 151. Publisher Full Text\n\nBavarsad Shahripour R, Hemati A, Hosseinzadeh MA: A randomized trial to assess the long-term safety of NeuroAiD among Caucasian patients with acute ischemic stroke. Chin. J. Integr. Med. 2014; 20(11): 812–817. PubMed Abstract \| Publisher Full Text\n\nHarandi AA, Abolfazli R, Hatemian A, et al.: Safety and Efficacy of MLC601 in Iranian Patients after Stroke: A Double-Blind, Placebo-Controlled Clinical Trial. Stroke Res. Treat. 2011; 2011: 1–5. Publisher Full Text\n\nYoung SH, Zhao Y, Koh A, et al.: Safety profile of MLC601 (Neuroaid) in acute ischemic stroke patients: A Singaporean substudy of the Chinese medicine neuroaid efficacy on stroke recovery study. Cerebrovasc. Dis (Basel, Switzerland). 2010; 30(1): 1–6. PubMed Abstract \| Publisher Full Text\n\nFabiaña NL, Chen CL, Venketasubramanian N, et al.: Sex and the treatment effect in the Chinese Medicine NeuroAiD Efficacy on Stroke recovery (CHIMES) trial. J. Clin. Neurosci. 2016; 33: 269–270. PubMed Abstract \| Publisher Full Text\n\nWidmann C, Gandin C, Petit-Paitel A, et al.: The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia. Sci. Rep. 2018; 8(1): 18062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoha Ou Maati H, Borsotto M, Chatelain F, et al.: Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation. Neuropharmacology. 2012; 63(4): 692–700. PubMed Abstract \| Publisher Full Text\n\nNavarro JC, Gan HH, Lao AY, et al.: Baseline characteristics and treatment response of patients from the Philippines in the CHIMES study. International journal of stroke: official journal of the International Stroke. Society. 2014; 9: 102–105. Publisher Full Text\n\nNavarro JC, Chen CL, Lagamayo PD, et al.: CHIMES-I: sub-group analyzes of the effects of NeuroAiD according to baseline brain imaging characteristics among patients randomized in the CHIMES study. Int. J. Stroke. 2013; 8(6): 491–494. PubMed Abstract \| Publisher Full Text\n\nVenketasubramanian N, Young SH, Tay SS, et al.: CHInese Medicine NeuroAiD Efficacy on Stroke Recovery - Extension Study (CHIMES-E): A Multicenter Study of Long-Term Efficacy. Cerebrovasc. Dis (Basel, Switzerland). 2015; 39(5-6): 309–318. PubMed Abstract \| Publisher Full Text\n\nVenketasubramanian N, Chen CL, Gan RN, et al.: A double-blind, placebo-controlled, randomized, multicenter study to investigate CHInese Medicine Neuroaid Efficacy on Stroke recovery (CHIMES Study). Int. J. Stroke. 2009; 4(1): 54–60. PubMed Abstract \| Publisher Full Text\n\nSuwanwela NC, Chen CLH, Lee CF, et al.: Effect of Combined Treatment with MLC601 (NeuroAiDTM) and Rehabilitation on Post-Stroke Recovery: The CHIMES and CHIMES-E Studies. Cerebrovasc. Dis (Basel, Switzerland). 2018; 46(1-2): 82–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CL, Venketasubramanian N, Lee CF, et al.: Effects of MLC601 on early vascular events in patients after stroke: the CHIMES study. Stroke. 2013; 44(12): 3580–3583. PubMed Abstract \| Publisher Full Text\n\nSiddiqui FJ, Venketasubramanian N, Chan ES, et al.: Efficacy and safety of MLC601 (NeuroAiD®), a traditional Chinese medicine, in poststroke recovery: a systematic review. Cerebrovasc. Dis (Basel, Switzerland). 2013; 35 Suppl 1: 8–17. PubMed Abstract \| Publisher Full Text\n\nGonzález-Fraile E, Martín-Carrasco M, Ballesteros J: Efficacy of MLC601 on functional recovery after stroke: A systematic review and meta-analysis of randomized controlled trials. Brain Inj. 2016; 30(3): 267–270. PubMed Abstract \| Publisher Full Text\n\nGandin C, Widmann C, Lazdunski M, et al.: MLC901 Favors Angiogenesis and Associated Recovery after Ischemic Stroke in Mice. Cerebrovasc. Dis (Basel, Switzerland). 2016; 42(1-2): 139–154. PubMed Abstract \| Publisher Full Text\n\nQuintard H, Borsotto M, Veyssiere J, et al.: MLC901, a traditional Chinese medicine protects the brain against global ischemia. Neuropharmacology. 2011; 61(4): 622–631. PubMed Abstract \| Publisher Full Text\n\nVenketasubramanian N, Kumar R, Soertidewi L, et al.: The NeuroAiD Safe Treatment (NeST) Registry: a protocol. BMJ Open. 2015; 5(11): e009866. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeurteaux C, Widmann C, Moha ou Maati H, et al.: NeuroAiD: properties for neuroprotection and neurorepair. Cerebrovascular diseases (Basel, Switzerland). 2013; 35 Suppl 1: 1–7. PubMed Abstract \| Publisher Full Text\n\nHeurteaux C, Gandin C, Borsotto M, et al.: Neuroprotective and neuroproliferative activities of NeuroAid (MLC601, MLC901), a Chinese medicine, in vitro and in vivo. Neuropharmacology. 2010; 58(7): 987–1001. PubMed Abstract \| Publisher Full Text\n\nLorivel T, Gandin C, Veyssière J, et al.: Positive effects of the traditional Chinese medicine MLC901 in cognitive tasks. J. Neurosci. Res. 2015; 93(11): 1648–1663. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenketasubramanian N, Lee CF, Young SH, et al.: Prognostic Factors and Pattern of Long-Term Recovery with MLC601 (NeuroAiD™) in the Chinese Medicine NeuroAiD Efficacy on Stroke Recovery - Extension Study. Cerebrovasc. Dis (Basel, Switzerland). 2017; 43(1-2): 36–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChankrachang S, Navarro JC, de Silva DA , et al.: Prognostic factors and treatment effect in the CHIMES study. J. Stroke Cerebrovasc. Dis. 2015; 24(4): 823–827. PubMed Abstract \| Publisher Full Text\n\nSidddiqi SA, Kamal AK: Use of traditional Chinese medication for post-stroke recovery. J. Pak. Med. Assoc. 2011; 61(9): 933–934. PubMed Abstract\n\nVenketasubramanian N, Lee CF, Wong KS, et al.: The value of patient selection in demonstrating treatment effect in stroke recovery trials: lessons from the CHIMES study of MLC601 (NeuroAiD). J. Evid. Based Med. 2015; 8(3): 149–153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandian JD, Liu M, Misbach J, et al.: Alternative Therapies for Stroke Treatment in Asia. Int. J. Stroke. 2011; 6(6): 541–543. Publisher Full Text\n\nLoo KW, Gan SH: Burden of stroke in the Philippines. Int. J. Stroke. 2013; 8(2): 131–134. Publisher Full Text\n\nVitaliano GD, Kim JK, Kaufman MJ, et al.: Clathrin-nanoparticles deliver BDNF to hippocampus and enhance neurogenesis, synaptogenesis and cognition in HIV/neuroAIDS mouse model. Commun. Biol. 2022; 5(1): 236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFung FY, Linn YC: Developing traditional chinese medicine in the era of evidence-based medicine: current evidences and challenges. Evid. Based Complement. Alternat. Med. 2015; 2015: 425037.\n\nBousser M-G: Drugs from Natural Substances: Why Study Them in Cerebral Infarction. Cerebrovasc. Dis. 2013; 35(Suppl. 1): IV–VI. PubMed Abstract \| Publisher Full Text\n\nNavarro JC, Chen CLH, Lee CF, et al.: Durability of the beneficial effect of MLC601 (NeuroAiD™) on functional recovery among stroke patients from the Philippines in the CHIMES and CHIMES-E studies. Int. J. Stroke. 2016; 12(3): 285–291. PubMed Abstract \| Publisher Full Text\n\nLee HP, Liu YC, Lin MF: Effects of Different Genres of Music on the Psycho-Physiological Responses of Undergraduates. Hu li za zhi J. Nurs. 2016; 63(6): 77–88. PubMed Abstract \| Publisher Full Text\n\nMortazavi N, Novin K, Bidari Zerehpoosh F, et al.: Intracyctic Papillary Carcinoma of the Breast: Report of a Rare Case and Literature Review.2017; 10(7): e7259.\n\nGhandehari K, Mood Z, Ebrahimzadeh S, et al.: NeuroAid (MLC601) versus piracetam in the recovery of post-infarct homonymous hemianopsia. Neural Regen. Res. 2011; 6: 418–422.\n\nLin DJ, Finklestein SP, Cramer SC: New Directions in Treatments Targeting Stroke Recovery. Stroke. 2018; 49(12): 3107–3114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShoshan-Barmatz V, Mizrachi D, Keinan N: Oligomerization of the mitochondrial protein VDAC1: from structure to function and cancer therapy. Prog. Mol. Biol. Transl. Sci. 2013; 117: 303–334. Publisher Full Text\n\nHuang S, Mingxia L, Xiaowei Pan XP, et al.: The potential of MLC901 (NeuroAiD II™), a traditional Chinese medicine. Neurosci. Res. Notes. 2019; 2: 18–24. Publisher Full Text\n\nHan S-Y, Hong Z-Y, Xie Y-H, et al.: Therapeutic effect of Chinese herbal medicines for post stroke recovery: A traditional and network meta-analysis. Medicine. 2017; 96(49): e8830. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaleh AY: Beyond stroke therapy, neuroaid (a chinese herbal) has an effect on cognition and neurogenesis, a bibliometric study. figshare. Figure. 2024. Publisher Full Text\n\nSaleh AY: PRISMA_2020_checklist_AYS. figshare. Dataset. 2024. Publisher Full Text\n\nvan Eck NJ , Waltman L: Citation-based clustering of publications using CitNetExplorer and VOSviewer. Scientometrics. 2017; 111(2): 1053–1070. Publisher Full Text" }	[ { "id": "305119", "date": "02 Aug 2024", "name": "Ping-Chung Leung", "expertise": [ "Reviewer Expertise Research on traditional medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStroke is a common debilitating disease with yet no standard treatment to improve the physical outcomes and mental disturbances during the rehabilitation period. Use of Herbal medicine is yet a controversial form of remedy not widely accepted. The use of substances of animal origin is another worrying component. Authors rightly completed a thorough search for the support of using Euro Aid. Apparently, most of the references originate from the CHIMES study. Authors could discuss, via the literature reviews, all the points raised 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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [ { "c_id": "12149", "date": "06 Aug 2024", "name": "ARMAN YURISALDI SALEH", "role": "Author Response", "response": "Thank you to the reviewers for their kindness in providing feedback and suggestions." } ] } ]	1	https://f1000research.com/articles/13-799
https://f1000research.com/articles/13-798/v1	16 Jul 24	{ "type": "Research Article", "title": "Early, typical, and late talkers: an exploratory study on predictors of language development in the first two years of life", "authors": [ "Maria Grazia Logrieco", "Ilaria Nicolì", "Maria Spinelli", "Francesca Lionetti", "Giulio D'Urso", "Giulia Carlotta Guerra", "Valeria D'Aloia", "Giusi Toto", "Mirco Fasolo", "Maria Grazia Logrieco", "Maria Spinelli", "Francesca Lionetti", "Giulio D'Urso", "Giulia Carlotta Guerra", "Valeria D'Aloia", "Giusi Toto", "Mirco Fasolo" ], "abstract": "Background The consensus in scientific literature is that each child undergoes a unique linguistic development path, albeit with shared developmental stages. Some children excel or lag behind their peers in language skills. Consequently, a key challenge in language acquisition research is pinpointing factors influencing individual differences in language development.\n\nMethods We observed children longitudinally from 3 to 24 months of life to explore early predictors of vocabulary size. Based on the productive vocabulary size of children at 24 months, 30 children met our sample selection criteria: 10 late talkers and 10 early talkers, and we compared them with 10 typical talkers. We evaluated interactive behaviors at 3, 6, 9 and 12 months, considering vocal production, gaze at mother’s face, and gestural production during mother-child interactions, and we considered mothers’ report of children’s actions and gestures and receptive-vocabulary size at 15 and 18 months.\n\nResults Results indicated early precursors of language outcome at 24 months identifiable as early as 3 months in vocal productions, 6 months for gaze at mother’s face and 12 months for gestural productions.\n\nConclusions Our research highlights both theoretical and practical implications. Theoretically, identifying the early indicators of belonging to the group of late or early talkers underscores the significant role of this developmental period for future studies. On a practical note, our findings emphasize the crucial need for early investigations to identify predictors of vocabulary development before the typical age at which lexical delay is identified.", "keywords": [ "late talkers", "early talkers", "typical talkers", "gender", "language development", "individual differences", "interactive behaviors", "gestures" ], "content": "1. Introduction\n\nThere is extraordinary variability in the vocabulary size of very young children (Bates, Bretherton & Snyder, 1991; Fasolo, D’Odorico, Costantini & Cassibba, 2010; Fenson, Dale, Reznick, Bates, Thal, Pethick, 1994; Goldfield, 1987; Hampson & Nelson, 1993; Huttenlocher, Haight, Bryk, Seltzer & Lyons, 1991). The scientific literature converges on the notion that every child follows his own path in linguistic development while sharing the same developmental stages with others, with some children standing out as stronger or weaker in their language skills than their peers. Indeed, a child of 24 months acquiring English or Italian with a typical development has a mean of 300 words in production (Brown, 1973; Caselli &-à Casadio, 2014), early talkers can have in their vocabulary up to 500 words at the same age, while late talkers tend to produce less than 50 words (Rescorla, 2002). One of the major challenges in the study of language acquisition is therefore to identify factors that can contribute to individual variation in language development. Considering the complexity of language development which is the joint product of maturation and experience (Carpenter, Nagell, Tomasello, Butterworth, & Moore, 1998; Tomasello, 1995) epidemiological studies have provided an integrative model of predictors. The model includes biological factors such as gen-der, child’s communication skills, and the quality of language stimulation a child receives from his parents as well as variables that may affect it, such as the level of education, and the socio-economic status (Oller, Eilers, Neal & Schwartz, 1999; Stanton-Chapman, Chapman, Bainbridge & Scott, 2002; Zambrana, Pons, Eadie, & Ystrom, 2014; Reilly et al., 2007; Eadie, Levickis, McKean, Westrupp, Bavin, Ware, Gerner, Reilly, 2022; Hoff, 2013). While there are several studies on the effect of environmental factors on language acquisition, few studies have focused on specific child’s behaviors that are detectable from the early months. Considering the role of gender in language skills variability, literature states that girls have generally superior linguistic abilities compared to age-matched boys (Zambrana et al., 2012; Huttenlocher et al., 1991). On the other hand, a recent meta-analysis pointed out that expressive language outcomes did not differ between boys and girls, although the variability among studies was significant (Fisher, 2017; Wallentin, 2020). For what pertains to the child’s communicative characteristics, research in literature has stated that a combination of early neurobiological and genetic factors, such as gender, and concurrent environmental variables and individual characteristics, such as verbal and non-verbal behaviors may have an effect on the slow language development (Schjølberg, Eadie, Zachrisson, Øyen, 2011; Zubrick, Taylor, Rice, Slegers, 2007) observable in the dyadic context already at 3 months. Indeed, longitudinal studies suggested that infant gaze at 10 months plays a quantifiable developmental role in children’s early linguistic and social–cognitive development (Brooks & Meltzoff, 2015). In this direction, research on preverbal pro-ductions emphasized that canonical bubbling at 10 months represents an important predictor to identify infants at extreme risk for speech and language disorders at the end of the first year of life (Oller, Eilers, Neal, and Schwartz, 1999). These studies provided important evidence on the investigation of early predictors of child later outcome. However, studies investigating predictors from the first few months onward use only parent report questionnaires to assess predictors while those using observational measures have considered language development starting from 10 months of age, thus, limiting our understanding of early candidate precursors. Following the multidimensional perspective suggested by prior works, in the present study we combined observational measures and parent report questionnaire, and we investigated the potential early markers of language development, including gender and communicative linguistic skills of the child arising from 3 months of age.\n\nTherefore, in the present study we involved children in different point of the language spectrum – late, typical, and early talkers - as young as 3 months old, following them up to 24 months of age. Specifically, the aim of our research is to explore if different social cues (Hollich, Hirsh-Pasek, Golinkoff, Brand, Brown, Chung, and Bloom, 2000) produced by the child since 3 months may be informative of the subsequent language outcome, that is the breadth of the productive vocabulary size at 2 years. Specifically, as social cues, we considered vocal production, gaze at mother’s face, actions and gestures, and receptive vocabulary size which represent the foundations for the later development of language (see McCathren, Yoder & Warren, 1999; Vihman, Macken, Miller, Simmons & Miller, 1985; Brooks & Meltzoff, 2005, 2008; Carpenter, Nagell, Tomasello, Butterworth & Moore, 1998; Bates, Thal & Marchman, 1991; Iverson & Thelen, 1999; Lyytinen, Eklund & Lyytinen, 2005; Thal, Marchman & Tomblin, 2013; Goldin-Meadow and Alibali, 2013). In addition, the role of gender was considered to potentially control for gender differences while exploring its contribution at the same time.\n\nLate talkers are children who show a vocabulary delay in the absence of any sensory, cognitive, or neurological deficit (Buschmann, Jooss, Rupp, Dockter, Blaschtikowitz, Heggen & Pietz, 2008; Rescorla & Lee, 2000; Silva, 1980; Whitehurst & Fischel, 1994). For the aim of the present study, we adopted the most common criteria found in the literature and focused on 24 months children who have barely reached a 50-word vocabulary and/or use no word combinations (Fenson, Dale, Reznick, Thal, Bates, Hartung & Reilly, 1994; Dollaghan, 2013; Rescorla, 2002). It is well known that late talkers are not a homogenous group in terms of their developmental outcomes: some catch up, as captured by the “late bloomers” metaphor and some will experience persistent learning difficulties (Chilosi et al., 2019). Furthermore, language delay has a multi-dimensional impact on learning (see, for example, Paul et al., 1997; Rescorla, 2002, 2005, 2009; Rescorla & Dale 2013) and socioemotional development (see, for example, Paul, 1991; Carson, Klee, Carson & Hime, 1997, 1998; Irwin, Carter & Briggs-Gowan, 2002). Hence, the early investigation of factors associated with a language delay is crucial for the early intervention so that education pro-grams can be implemented to maximize positive child developmental outcomes across a variety of domains (see, for example, Buschmann et al., 2015; Roberts & Kaiser, 2015; St Clair et al., 2011). Although there is a significant interstudy variability in the analysis of language development in late talkers (Fisher, 2017), research findings have been able to identify a series of specific communicative and linguistic characteristics of late talkers. More specifically, different authors have shown that late talkers have a narrow phonological inventory of both vowels and consonants, mostly characterized by simple syllabic structures, unlike typical talkers of the same age (Paul & Jennings, 1992; Rescorla & Ratner, 1996; Mirak & Rescorla, 1998; Carson et al., 2003). Interestingly, differences between late and typical talkers are not strictly limited to linguistic variables. In fact, empirical studies have suggested that some communicative behaviors of late talkers, such as the different degree in gestural production, represented potential early predictors of delay in subsequent language development (Ellis & Thal, 2008; Thal et al., 2013). Studies on gestural communication in children with linguistic delay highlighted significant differences at 20 months between late and typical talkers, resulting in a disadvantage of late talkers in the production of gestures and the combination of gesture and gaze at mother’s face (Fasolo & D’Odorico, 2002). In the same perspective, we aim to explore the communicative behaviors of late and typical talkers, but investigating instead, the predictors of language development from the first months of life. Moreover, we do not limit our examination to typical vs. late talkers, as traditionally done in the extant literature. Indeed, with the aim of better identifying what language development predicts more broadly, we involve into our analysis early talkers too. In terms of productive vocabulary, early talkers are children who scored around the 90th percentile based on parent report of toddler’s expressive language (Fenson et al., 1994). Research has shown that early spoken language development can play a substantial role in oral and written language performance (Lyytinen, Poikkeus, Laakso, Eklund & Lyytinen, 2001; Lyytinen, Ahonen, Eklund, Guttorm, Kulju, Laakso & Viholainen, 2004a, b; Scarborough and Dobrich, 1990). Indeed, there are several studies on the early talkers regarding their learning outcomes during school-age years (see, for example, Crain-Thoreson & Dale 1992; Lyytinen et al., 2001, 2004a, b; Scarborough and Dobrich, 1990). However, to the best of our knowledge, no study has investigated the communicative behaviors of early talkers at the initial stage of language development.\n\nTo sum up, there has been an increase in studies interested in identifying communicative and linguistic factors related to the variability of the child’s expressive language. However, most of these studies involved children older than 10 months of age. Based on the literature we have identified two categories of predictors that can be fruitfully investigated starting from the first months of life and can be considered key competences underlying the subsequent productive vocabulary size (McCathren, Yoder & Warren, 1999; Vihman, Macken, Miller, Simmons & Miller, 1985; Brooks & Meltzoff, 2005, 2008; Carpenter, Nagell, Tomasello, Butterworth & Moore, 1998; Bates, Thal & Marchman, 1991; Iverson & Thelen, 1999; Lyytinen, Eklund & Lyytinen, 2005; Thal, Marchman & Tomblin, 2013; Goldin-Meadow and Alibali, 2013). With direct observational measures we explored the number of vocal behaviors, gaze at mother’s face and gestures produced by the child in interaction with the caregiver. Moreover, we evaluated actions, gestures and the receptive vocabulary size using the “Primo Vocabolario del Bambino (PVB)” questionnaire (Caselli, Bello, Rinaldi, Stefanini, & Pasqualetti, 2015) that is the Italian version of the MacArthur-Bates CDI (Fenson, Marchman, Thal, Dale, Reznick, Bates, 2007) filled out by parents. We have defined these predictors as interactive behaviors, as all these variables emerged in the social scene between child and adult (Tomasello, 1995). Besides, we considered the role of gender to control for its potential effect, while also exploring its interplay and contribution with the set of interactive behaviors considered. Given the relatively small sample size and the explorative nature of the current study, we adopted a descriptive approach. We aim to investigate, between the 3rd and the 18th month of age, which interactive behaviors best predicted the probability to be at 24 months old a typical, late, or an early talker, and at which time point this may occur. If early intervention is to be provided in the critical period for language development, it is necessary to investigate and identify early predictors in younger children as early as possible. Hence the goal of the present study is to add information on an evolutionary period sparsely considered in the extant literature, and that is the study of developmental trajectories of early and late talkers starting from early months.\n\n\n2. Methods\n\nThis study included 30 monolingual Italian mother–child dyads. The dyads were selected from a larger and longitudinal study on parenting and infant development in Italy at University of Chieti before the lockdown due to Covid-19. The vocabularies of 10 late talkers (LT), 10 typical talkers (TT) and 10 early talkers (ET) were selected out of a pool of 80 MB-CDI forms (Caselli, Bello, Rinaldi, Stefanini, & Pasqualetti, 2015) completed by parents for children (42 males and 38 females) aged 24 months. Specifically:\n\n- LT (9 males and 1 female) produced less than 50 words (M = 37; range: 15-59), corresponding to the 10th percentile of productive vocabulary size,\n\n- ET (2 males and 8 females) produced more than 500 words (M = 562, range: 509-640), corresponding to the 90th percentile of productive vocabulary size,\n\n- TT (4 males and 6 females) produced around 225 words (M = 290, range: 222-350), and their vocabulary size was closer to the 50th normal mean value.\n\nMother’s mean age was 34.20 (SD = 5.35). Concerning socioeconomic status, 80 % of them had a medium economic status, and 20% had a low economic income (LT: 70% medium income; ET: 80% medium income; TT: 90% medium income). In relation to educational level, 66.6% of mothers had a high school degree or less, and 33.3% a bachelor’s or master’s degree (LT: bachelor’s degree 40%, high school degree 50%, secondary school degree 10%; ET: bachelor’s degree 30%, high school degree 60%, secondary school degree 10%; TT: bachelor’s degree 30%, high school degree 70%).\n\nParticipants were recruited at an Italian urban hospital within 2 days from the child’s birth, and mothers were asked to join a research project on infant development. The mothers who agreed to be part of the research study were then contacted 1 to 2 months later and invited to come to the laboratory of Developmental Psychology. Only mothers gave us their willingness to participate to the experiment in lab. A written informed consent (approved by the ethical committee) for participation in the study has been obtained by the mothers. Before testing, each mother–child dyads participated in a familiarization phase in a room next to the proper observational room, equipped with age-specific toys. The mother-child dyads were observed during a video-recorded semi-structured play session conducted in the laboratory when the child was 3, 6, 9 and 12 months old. At 3 and 6 months, the mother and the infant were observed during a video-recorded interaction session. The mother sat in a straight-backed chair without arms and was asked to “play and talk to your baby as you would at home”. The child was in front of the mother sitting on a bouncer at 3 months and on a highchair at 6. The sessions were videotaped. The camera was controlled by an experimenter present in the room and placed behind the mother. It framed both the infant’s face and mother’s face, thanks to a mirror positioned behind the infant’s station. The total session lasted approximately 4 minutes. At 9 and 12 months, mother and child were filmed while playing with a standard set of toys. The age-appropriate toy set represented objects with which all children were familiar in their everyday routines and were provided with the specific aim of stimulating communicative and interactive exchanges. At 9 months, at the beginning of the interaction, we provided cubes to build a tower, while in the middle of the interaction we provide a book with illustrations of animals. At 12 months, we provided an illustrated booklet with figures of animals and a set of toy food. The sessions were videotaped. The camera was controlled by an experimenter present in the room with a camera placed in front of the mother and the child. The interaction lasted approximately 6 minutes.\n\nAt 15-, 18- and 24-months, the parents filled the questionnaire MB-CDI. The “Word and Gesture – WG” version was administered at 15 months, while the “Words and Sentences – WS” version at 18 and 24 months.\n\n2.3.1 Interactive behaviors in the first year of life\n\nTwo trained transcribers, working independently one from the other and using CHAT (CHILDES system) format (MacWhinney, 2000), transcribed all the interactive behaviors at 3, 6, 9 and 12 months directly from the mother – child recorded interaction. The second transcriber was not informed of the subject’s group assignment until the transcription was completed. The interactive behaviors were coded with a multi-dimensional schema:\n\nVocal productions. Each production was counted as a single unit of analysis when separated by at least one second. Crying vocalizations and sounds of discomfort were not considered. The frequency of the child’s vocal productions per minute was calculated. Child’s vocal productions were coded as follows: vocalization (vowel sound typically produced open-mouthed), grunt (consonant sound produced by closed or semi closed mouth), onomatopoeia (sounds that reproduce the sound of animals, or the noise produced by vehicles or actions), babbling (syllabic sound consisting of a consonant and a vowel), proto words (sounds similar to words that take on specific meaning in certain contexts).\n\nGaze at mother’s face. The unit of analysis corresponds to a child’s gaze at mother’s face. Every time that the child looked at the mother’s face, the gaze was coded. Gaze at the mother’s body, hair, hands, and gaze to environment was not considered. The frequency of child’s gaze at mother’s face per minute was calculated.\n\nGestural productions. The unit of analysis corresponded to a child gestural production. Every gesture accompanied by other behaviors that signal the communicative intent (i.e., direct gaze to the interlocutor) was coded. The frequency of child gestural productions per minute was calculated. Gestural productions included deictic gestures: pointing (extending the index finger in the direction of an object, an event, or a person), giving (put an object towards the hand of the interlocutor), showing (put the object along the line of sight of the interlocutor). Moreover, we included representative gestures: iconic gestures (gestures which refer to objects, persons, or events, reproducing physical or functional characteristics), conventional (gestures with a culturally defined meaning and form e.g., waving bye, asking for silence pressing the index finger against the lips).\n\n2.3.2 Reliability\n\nTranscription reliability was calculated on 25% of the total videos. Point-by-point interactive behaviors comparisons were made, and the intercoder reliability ranged from 0,80 to 0,95. To maintain data consistency, only the material produced by the first transcriber was used for the final analysis. Inter-rater reliability for each predictor was computed by having a second rater that independently scored the subject performance on each measure. The inter-rater reliability was coded on 10% of the sample; the inter-coder reliability ranged from 0.83 to 0.94. Disagreements were solved with discussion to identify a unique score.\n\n2.3.3 Interactive behaviors in the second year of life\n\nThe interactive behaviors in the second year of life at 15-, 18- and 24-months were assessed using the scores assigned by parents at the CDI. Specifically, we considered:\n\nCDI Actions and Gestures. We considered the number of actions and gestures produced by the child reported by the mother at 15 months. This category included communicative gestures, games and routines, actions with objects, symbolic game (i.e., pretending to be mom or dad, imitating the actions of the adult, and playing pretend with objects). Data was missing for a male in the LT group for CDI actions at 15 months.\n\nCDI Productive and Receptive Vocabulary Size. We considered the receptive vocabulary size of the child (the number of words understood by the child) as reported by the mother at 15 and 18 months. At 24 months, we considered the productive vocabulary size (the number of words produced by the child) which was used as a criterion for inclusion in one of the 3 groups (late, typical, and early talkers). The CDI Receptive vocabulary measure at 18 months was missing for two children in the LTs group (1 male, 1 female) and one female in the ETs group.\n\nFor the analysis of data, given the relatively small sample size, we avoided adopting a Null-Hypothesis Significance Testing Approach, rather we commented on the effect sizes describing patterns of findings (Wasserstein et al., 2019). After having explored mean and standard deviation values as well as bivariate correlations among study variables, we ran and compared a series of multinomial logistic regression models including group membership (early, typical, and late talkers) as the outcome variable, and as predictors vocal production, gaze at mother’s face, gesture production, CDI actions and gestures, and CDI receptive vocabulary at the different time points investigated. We included one predictor at time in the regression models, to explore the differential effect of each variable across the different time points considered. As vocal production, gaze at mother’s face and gesture production were collected across multiple time points, we used Akaike Weights to identify what time point was more relevant for each specific variable. Akaike Weights represent a measure of the strength of adaptation for each model and the probability, conditioned to the set of models considered, that the same model can predict new data (McElreath, 2016; Vandekerckhove Matzke & Wagenmakers, 2015; Wagenmakers & Farrell, 2004). Akaike weights range from 0 to 1 and the higher the value, the more the model comparatively describes the data accurately. In each multinomial regression model, as groups were unbalanced regarding gender, we included gender as a predictor to simultaneously control for its impact as well as exploring its contribution. As the sample was relatively homogenous in terms of socio-economic and educational background, we did not include any other control variable in the model. Linear predicting probabilities of belonging to a specific group as a function of the predictors considered at different time points were plotted and graphically explored (Figure 1-5). Finally, as a follow-up analysis, for the best model selected we provided the probability of belong to a specific group for low (< 25°), medium (< 25° < 75°) and high (> 75°) levels of the predictor. All analyses were run with the statistical software R version 4.2.3, package ‘nnet (Ripley, Venables & Ripley, 2016) for multinomial regression analysis and gglpot2 (Wickham, 2016) for the graphical representation of findings.\n\n\n3. Results\n\nMeans, SDs, and correlation values among variables of interest are reported in Table 1 and Table 2.\n\nAkaike Weights derived from the comparison of multinomial regression models, as reported in Table 3, suggested that early vocal production at 3 months of age was more able to predict subsequent 24 months group membership than vocal production at 6, 9, and 12 months. In relation to gaze at mother, the model receiving most support was the one including gaze at 6 months, suggesting that gaze at the mother in this specific developmental period was more predictive of language development than not gaze at 3, 9, and 12 months of age. Regarding the gestural production, its assessment at 12 months was more predictive of group membership than gestural production at 9 months. Hence, vocal production at 3 months, gaze at mother at 6 months, gestural production at 12 months comparatively emerged as the most informative markers of later vocabulary productions, and hence group membership, at 2 years of age. A more in-depth analyses of findings for each predictor is reported in the paragraphs below.\n\n3.2.1 The role of child vocal production\n\nAs evidenced above, vocal production at 3 months of age was more predictive of group membership at 24 months compared to vocal production at months 6, 9 and 12. This result suggests that vocal productions that occur early in life are potentially first key predictors of verbal productivity at 24 months. More specifically, the graphical exploration of findings (see Figure 1) showed that the higher the vocal production at 3 months of age, the lower was the probability of belong to the LTs group with a stronger effect for males (see Figure 1) – which were still predominant in the LTs group. Predictive probabilities of belonging to each group for low (below the 25th percentile), medium (between the 25th and 75th percentile) and high (above the 75th percentile) levels of vocal production at 3 months (Table 4). The pattern identified at 3 months of age was comparable to that at 12 months, though – coherently with the fact that this model was less supported by Akaike weights - the effect was less strong. At 9 months of age, vocal production was not predictive of group membership while at 6 months the higher the vocal production, the higher the probability for females to be in the ET group. No predictive effect for LT belonging was evident.\n\n3.2.2 The role of gaze at mother’s face\n\nConcerning the role of gaze at mother Akaike weights (Table 3) suggested that a critical time point was 6 months, as depicted in Figure 2. Predicting probabilities of group membership for low, medium, and high values of gaze at mother’s face at 6 months are reported in the supplementary material. As it was for vocal reactivity at 3 months, the higher the number of gazes at mother at 6 months of age, the lower was the probability of belonging to the LTs group with the strongest effect for males. A comparable pattern emerged at 3 months, though with a less strong effect. At 9 and 12 months, gaze at mother’s face seems progressively less significant in predicting group membership, for both males and females. Hence, results suggest that gaze at mother at 6 months of age comparatively represents a better predictor (compared to gaze at mother at the other investigated time points) of language productivity at 24 months, and it might particularly help in identifying males that are more likely to become late talkers. The probability values of belonging to a group was then explored for the low, medium, and high values of the gaze at mother’s face at 6 months (Table 5).\n\n3.3.3 The role of child gestural production\n\nFor what pertains to gestural production, at 9 months few frequencies other than 0 emerged in the production of gestures, and the frequency per minute of gestures at 9 months seems to be relatively insignificant in predicting the group membership. A key time point for the prediction of group membership was age 12 months (see Table 3 and Figure 3). In males, as the frequency per minute of gestures produced increases, the probability of belonging to the group of late talkers decreases. For both males and females, the higher the gesture production at 12 months, the higher was the probability to be an early talker. As for the other variables, the probability values of belonging to a group was then explored for the low, medium, and high values of the gestural production at 12 months, (Table 6).\n\n3.3.4 The role of CDI actions and gestures\n\nIn relation to actions and gestures, (Figure 4), results suggested that the higher the number of gestures and actions at 15 months, the higher the probability of being in the ETs, and the lower the probability of being in the LTs, with a comparable trend for males and females. The probability values of belonging to a group was explored for the medium and high values of the actions and gestures model at 15 months (Table 7).\n\n3.3.5 The role of CDI receptive vocabulary size\n\nFinally, we investigated the role of receptive vocabulary at 15 and 18 months. As reported in the plot of predicted probabilities (Figure 5), as the receptive-vocabulary size increases, the probability of being ETs increases too, with a similar trend for both male and female, while the probability of belonging to TTs and LTs decreases. The probability analysis of receptive vocabulary size is in Table 8. The probabilities of belonging to the group for low, medium, and high values of the variables of interest were then explored, considering the mean of the receptive-vocabulary size at 15 and 18 months when possible, or only one time point if data were available for only one assessment phase (Figure 6).\n\nNote: V is the abbreviation of Vocal at 3, 6, 9 and 12 months; G is the abbreviation of Gaze at 3, 6, 9 and 12 months, GE is the abbreviation of gestural production at 9 and 12 months, AG is the abbreviation of MB-CDI at 15 months, RV is the abbreviation of MB-CDI Receptive Vocabulary size at 15 and 18 months, and PV is the abbreviation of MB-CDI Productive Vocabulary size at 24 months.\n\n\n4. Discussion\n\nThe purpose of the study was to explore early predictors of the probability to be a late, typical, or early talker. In doing this, we investigated age points never considered in the prior literature. We specifically focused on a series of interactive behaviors, analyzed in the context of the parent-child interaction, as potential predictors. Moreover, we explored the contribution of gender. Our findings suggest that the roots of variability in the expressive vocabulary size, that can be reliably assessed when children are two years old, can be found very early in life, in infants as young as 3 months of age. More specifically, we found that vocal production at 3 months of age can comparatively be more able to predict subsequent language development than vocal productions later, and that the gaze at the caregiver’s face at 6 months can predict group membership better than gaze at the caregiver’s face later. Interestingly, even if based on a relatively small sample, the pattern of findings identified was consistent with the theoretical expectations. Specifically, that the higher was the frequency of the specific interactive behaviors considered, the higher was the likelihood to belong to an early talker vs. a typical or later talker’s condition. This study, although preliminary and explorative in nature, highlights the complexity that characterizes development. In line with what has been conceptualized as a “cognitive cascade”, in which elementary skills are the basis of more complex ones (Rose, Feldman, Jankowski & Van Rossem, 2008), language development also follows a “cascade effect”, in which early communicative skills are the basis of more advanced abilities (Fasolo, D’odorico, Costantini & Cassibba, 2010). Overall, our results emphasize that human language must be seen as structurally related to non-linguistic behaviors (Vihman, Macken, Miller, Simmons & Miller, 1985). In fact, from the earliest months the newborn produces simple sounds (Locke, 1989). Child’s vocal productions generate immediate and regular consequences in the social environment and gradually become more complex thanks to neurobiological maturation and the continuous interactions with the caregiver (Desmarais, Sylvestre, Meyer, Bairati, & Rouleau, 2008). The exercise in prelinguistic productions expands the repertoire of sounds and sequences of sounds to which the first words are associated with (Locke, 1989). Therefore, it is possible that at an early age the initial vocal production could have a “cascade effect” (Fasolo et al., 2010) on the more complex competences expressed at two years. The gaze at caregiver’s face at 6 months is the variable that better predicts the later language outcome. Indeed, the selective attention to the social scene, such as the attention to the caregiver’s face, is positively correlated with language development (Brooks & Meltzoff, 2005, 2008; Carpenter, Nagell, Tomasello, Butterworth & Moore, 1998). These findings suggest that the pre-linguistic production and the gaze at mother’s face, can be considered an expression of a “communicative instinct” that changes over time, and which unfolds in the face-to-face exchange between the infant and the caregiver (Tomasello & Kruger, 1992). When approaching the first year of life, children begin to use their motor skills for social, thus producing representational and deictic gestures that are aimed at communicating intentions and meanings (Aureli, Spinelli, Fasolo, Garito, Perucchini, & D’Odorico, 2017). In line with existing literature, our study underlies that the production of gestures at 12 and actions and gestures at 15 months is a predictor of the productive vocabulary size at two years (Iverson & Goldin-Meadow, 2005). The different degree of use of actions and gestures may be representative of a different degree of maturity of symbolic function, which is the underlying ability of the language development process (Bates et al., 1979). It is supposable that the competences, that children exercise with the production of actions and gestures during social interactions, associated with several developments in other areas (e.g., cognitive maturity) and parent’s responsiveness (Aureli, Spinelli, Fasolo, Garito, Perucchini & D’Odorico, 2017; Tomasello & Kruger, 1992) can influence the ability to attribute symbolic autonomy to a word. This ability is considered by some authors as fundamental in determining the transition from the first to the second lexical phase which determines the rapid increase in the productive vocabulary size (Bates et al., 1979). Regarding gender, consistent with data in the literature, our study found that gender appears to contribute to differences in language outcomes at 24 months (Huttenlocher, 1991). Specifically, females in our study seem to be more advantaged than males and were more likely to belong to the group of early or typical talkers, especially in the first year of life. It is important to specify that the conclusions on this outcome should be considered with caution. First, the imbalance between males and females is a question that does not allow us to generalize the data. Second, it is not possible to assume whether the differences are due to individual variables, such as gender, or environmental variables, such as the quantity or quality of input to which children are exposed.\n\n\n5. Conclusions\n\nThe study investigates linguistic trajectories of children at the opposite side of language spectrum - late and early talkers – starting from 3 months. We used a longitudinal de-sign, and we adopted an observational method. However, findings should also be considered in the light of some limitations. First, even if the sample size was noteworthy given the longitudinal design and the use of observational and laboratory-based measures, for reaching more conclusive research findings, a bigger sample is needed. Stated this, it is important to note that our sample is comparable to that adopted in other studies on late and early talkers (Fisher, 2017). Specifically, the percentage of late talkers we identified is consistent with empirical research and data reported in the international literature (Bello, Olioso, Anghinoni, Gavalotti & Caselli, 2014; Horwitz, Irwin, Briggs-Gowan, Heenan, Mendoza & Carter, 2003; Zubrick, Taylor, Rice, Slegers, 2007). A second limit is related to the unbalanced gender distributions. However, the number of males and females that we identified in the group of early and late talkers is coherent with data reported in the literature about early gender differences, emphasizing a language advantage in females (Doran, 1907; Nelson, 1973; Reznick & Goldfield, 1989). A third limit is that the interactive behaviors referring to the second year of life (CDI number of actions and gestures and CDI receptive-vocabulary size) were based exclusively on parent-report. In future studies, direct observational measures should be included at all time points, to evaluate interactive behaviors both during the first and second year of life. Finally, this study must be contextualized to the specific background in which the data have been collected, involving children from a single Country and with the same ethnic background. Future studies should consider the involvement of a larger group of children with different ethnic and socio-cultural backgrounds.\n\nNotwithstanding these limitations, we believe that our study points out the importance of the identification of communicative predictors of the expressive vocabulary starting from very early in life. Our study points out potential theoretical and applied implications. From a theoretical perspective, the early identification of the roots of belonging to the group of late or early talkers, underlie the main role of this evolutionary period in future studies. On the other hand, from an applied point of view, our results specify and reinforce the importance of an early investigation in order to identify the predictors of vocabulary development prior to the age point at which lexical delay is usually identified.\n\nThe early identification of children that might be more at risk of a delay in language acquisition can enable access to early intervention and education programs that maximize positive child developmental outcomes across a variety of domain. Importantly, because all investigated communicative indices were observed in a dyadic context, these results suggest that the parent-child dyad could be the target of early intervention and prevention programs. All the interactive behaviors considered imply skills that promote interactive exchange, confirming once again that language develops primarily in the context of face-to-face interaction between the infant and the caregiver. A key question for future studies could be the investigation of the effects of the caregiver’s communicative and linguistic competencies on the children linguistic path.\n\n\nEthics and consent\n\nThe study was approved by the ethical committee of the Department of Neuroscience Imaging and Clinical Science of the University of Chieti-Pescara (Ethical approval number: DNISC2962, 06.11.2019) and was conducted according to the American Psychological Association guidelines in accordance with the 1964 Helsinki Declaration.\n\nA written informed consent (approved by the ethical committee) for participation in the study has been obtained by the mothers.", "appendix": "Data availability statement\n\nDryad: Early, typical, and late talkers: an exploratory study on predictors of language development in the first two years of life. https://doi.org/10.5061/dryad.wwpzgmsrb (Logrieco, 2024a).\n\nThis project contains the following underlying data:\n\n• LateEarlyTypical_Talker.xlsx. Raw data file which includes the child’s data.\n\n• read_me_.docx. An explanatory document.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAureli T, Spinelli M, Fasolo M, et al.: The pointing–vocal coupling progression in the first half of the second year of life. Infancy. 2017; 22(6): 801–818. Publisher Full Text\n\nBates E, Bretherton I, Snyder LS: From first words to grammar: Individual differences and dissociable mechanisms. Vol. 20. . Cambridge University Press; 1991.\n\nBates JE, Freeland CAB, Lounsbury ML: Measurement of infant difficultness. Child Dev. 1979; 50:794–803. PubMed Abstract \| Publisher Full Text\n\nBello A, Remi L, Olioso G, et al.: Un’esperienza di screening per l’identificazione di bambini con ritardo di linguaggio nella provincia di Mantova: primi risultati e prospettive future. Psicol. Clin. Svilupp. 2014; 18(2): 329.\n\nBrooks R, Meltzoff AN: The development of gaze following and its relation to language. Dev. Sci. 2005; 8(6): 535–543. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooks R, Meltzoff AN: Infant gaze following and pointing predict accelerated vocabulary growth through two years of age: A longitudinal, growth curve modeling study. J. Child Lang. 2008; 35(1): 207–220. PubMed Abstract \| Publisher Full Text\n\nBrooks R, Meltzoff AN: Connecting the dots from infancy to childhood: A longitudinal study connecting gaze following, language, and explicit theory of mind. J. Exp. Child Psychol. 2015; 130: 67–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown R: A first language: The early stages. Harvard U. Press; 1973.\n\nBuschmann A, Jooss B, Rupp A, et al.: Children with develop-mental language delay at 24 months of age: results of a diagnostic work up. Dev. Med. Child Neurol. 2008; 50(3): 223–229. PubMed Abstract \| Publisher Full Text\n\nBuschmann A, Multhauf B, Hasselhorn M, et al.: Long-term effects of a parent based language intervention on language outcomes and working memory for late talking toddlers. J. Early Interv. 2015; 37(3): 175–189. Publisher Full Text\n\nCarpenter M, Nagell K, Tomasello M, et al.: Social cognition, joint attention, and communicative competence from 9 to 15 months of age. Monogr. Soc. Res. Child Dev. 1998; 63: i–174. PubMed Abstract \| Publisher Full Text\n\nCarson CP, Klee T, Carson DK, et al.: Phonological profiles of 2-year-olds with delayed language development. Am. J. Speech Lang. Pathol. 2003; 12: 28–39. PubMed Abstract \| Publisher Full Text\n\nCarson DK, Klee T, Perry CK, et al.: Measures of language proficiency as predictors of behavioral difficulties, social and cognitive development in 2 -year-old children. Percept. Mot. Skills. 1997; 84(3): 923–930. PubMed Abstract \| Publisher Full Text\n\nCarson DK, Klee T, Perry CK, et al.: Comparisons of children with delayed and normal language at 24 months of age on measures of behavioral difficulties, social and cognitive development. Infant Mental Health Journal: Official Publication of The World Association for Infant Mental Health. 1998; 19(1): 59–75. Publisher Full Text\n\nChuttenlocher J, Haight W, Bryk A, et al.: Early vocabulary growth: relation to language input and gender. Dev. Psychol. 1991; 27(2):236–248. Publisher Full Text\n\nMcCathren RB, Yoder PJ, Warren SF: The relationship between prelinguistic vocalization and later expressive vocabulary in young children with developmental delay. J. Speech Lang. Hear. Res. 1999; 42(4): 915–924. PubMed Abstract \| Publisher Full Text\n\nCaselli MC, Bello A, Rinaldi P, et al.: Il primo vocabolario del bambino: gesti, parole e frasi. Forme Lunghe e Forme Brevi del Questionario e Valori di Riferimento per la Fascia. 2015; pp. 8–36.\n\nChilosi AM, Pfanner L, Pecini C, et al.: Which linguistic measures distinguish transient from persistent language problems in Late Talkers from 2 to 4 years? A study on Italian speaking chil-dren. Res. Dev. Disabil. 2019; 89: 59–68. PubMed Abstract \| Publisher Full Text\n\nCrain-Thoreson C, Dale PS: Do early talkers become early readers? Linguistic precocity, preschool language, and emergent literacy. Dev. Psychol. 1992; 28(3): 421–429. Publisher Full Text\n\nDesmarais C, Sylvestre A, Meyer F, et al.: Systematic review of the literature on characteristics of late-talking toddlers. Int. J. Lang. Commun. Disord. 2008; 43(4): 361–389. PubMed Abstract \| Publisher Full Text\n\nDollaghan C: Late talker as a clinical category: A critical evaluation. Late talkers: Language development, interventions, and outcomes. 2013; 91–112.\n\nDoran EW: A study of vocabularies. The Pedagogical Seminary. 1907; 14(4): pp. 401–438. Publisher Full Text\n\nEadie P, Levickis P, McKean C, et al.: Developing Preschool Language Surveillance Models-Cumulative and Clustering Patterns of Early Life Factors in the Early Language in Victoria Study Co-hort. Front. Pediatr. 2022; 10: 10. Publisher Full Text\n\nEllis EM, Thal DJ: Early language delay and risk for language impairment. Perspectives on Language Learning and Education. 2008; 15(3): 93–100. Publisher Full Text\n\nFasolo M, D’Odorico L: Comunicazione gestuale nei bambini con sviluppo del linguaggio rallentato: una ricerca longi-tudinale. Psicol. Clin. Svilupp. 2002; 6(1): 119–138.\n\nFasolo M, D’odorico L, Costantini A, et al.: The influence of biological, social, and developmental factors on language acquisition in pre-term born children. Int. J. Speech Lang. Pathol. 2010; 12(6): 461–471. PubMed Abstract \| Publisher Full Text\n\nFenson L, Dale PS, Reznick JS, et al.: Variability in early communicative de-velopment. Monogr. Soc. Res. Child Dev. 1994; 59: i. Publisher Full Text\n\nFenson L, Marchman VA, Thal DJ, et al.: MacArthur–Bates Communicative Development Inventories: User’s Guide and Technical Manual. 2nd ed.Baltimore: Brookes; 2007.\n\nFisher EL: A systematic review and meta-analysis of predictors of expressive language outcomes among late talkers. J. Speech Lang. Hear. Res. 2017; 60(10): 2935–2948. PubMed Abstract \| Publisher Full Text\n\nGoldfield BA: The contributions of child and caregiver to referential and expressive language. Appl. Psycholinguist. 1987; 8(3): 267–280. Publisher Full Text\n\nGoldin-Meadow S, Alibali MW: Gesture’s role in speaking, learning, and creating language. Annu. Rev. Psychol. 2013; 64: 257–283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHampson J, Nelson K: The relation of maternal language to variation in rate and style of language acquisition. J. Child Lang. 1993; 20(2): 313–342. PubMed Abstract \| Publisher Full Text\n\nHoff E: Interpreting the early language trajectories of children from low-SES and language minority homes: implica-tions for closing achievement gaps. Dev. Psychol. 2013; 49(1): 4–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHollich GJ, Hirsh-Pasek K, Golinkoff RM, et al.: Breaking the language barrier: An emergentist coalition model for the origins of word learning. Monogr. Soc. Res. Child Dev. 2000; I 135. Publisher Full Text\n\nHorwitz SM, Irwin JR, Briggs-Gowan MJ, et al.: Language delay in a com-munity cohort of young children. J. Am. Acad. Child Adolesc. Psychiatry. 2003; 42(8): 932–940. PubMed Abstract \| Publisher Full Text\n\nHuttenlocher J, Haight W, Bryk A, et al.: Early vocabulary growth: relation to language input and gender. Dev. Psychol. 1991; 27(2): 236–248. Publisher Full Text\n\nIrwin JR, Carter AS, Briggs-Gowan MJ: The social-emotional development of “late talking” toddlers. J. Am. Acad. Child Adolesc. Psychiatry. 2002; 41(11): 1324–1332. PubMed Abstract \| Publisher Full Text\n\nIverson JM, Goldin-Meadow S: Gesture paves the way for language development. Psychol. Sci. 2005; 16(5): 367–371. PubMed Abstract \| Publisher Full Text\n\nIverson JM, Thelen E: Hand, mouth and brain. The dynamic emergence of speech and gesture. J. Conscious. Stud. 1999; 6(11-12):19–40.\n\nLocke JL: Babbling, and early speech: Continuity and individual differences. First Lang. 1989; 9(6): 191–205. Publisher Full Text\n\nLogrieco MGM: Early, typical, and late talkers: an exploratory study on predictors of language development in the first two years of life. [Dataset]. Dryad. 2024a. Publisher Full Text\n\nLogrieco MGM: Early, typical, and late talkers STROBE Checklist.2024b, May 3. Retrieved from osf.io/hfx5s. Publisher Full Text\n\nLyytinen H, Ahonen T, Eklund K, et al.: Early development of chil-dren at familial risk for Dyslexia follow-up from birth to school age Dyslexia.2004a; 10(3): 146–178.\n\nLyytinen H, Aro M, Eklund K, et al.: The development of children at familial risk for dyslexia: Birth to early school age. Ann. Dyslexia. 2004b; 54(2): 184–220. PubMed Abstract \| Publisher Full Text\n\nLyytinen P, Eklund K, Lyytinen H: Language development and literacy skills in late-talking toddlers with and without familial risk for dyslexia. Ann. Dyslexia. 2005; 55:166–192. PubMed Abstract \| Publisher Full Text\n\nLyytinen P, Poikkeus AM, Laakso ML, et al.: Language development and symbolic play in chil-dren with and without familial risk for dyslexia. J. Speech Lang. Hear. Res. 2001; 44: 873–885. PubMed Abstract \| Publisher Full Text\n\nMacWhinney B: The CHILDES Project: tools for analyzing talk. 3rd ed.Mahwah: Lawrance Erlbaum; 2000.\n\nMcElreath R: Statistical rethinking: A Bayesian course with examples in R and Stan. CRC Press; 2016.\n\nMirak J, Rescorla L: Phonetic skills and vocabulary size in late talkers: Concurrent and predictive relationships. Appl. Psycholinguist. 1998; 19(1): 1–17. Publisher Full Text\n\nNelson K: Structure and strategy in learning to talk. Monogr. Soc. Res. Child Dev. 1973; 38(2, Serial No. 149): 1. Publisher Full Text\n\nOller DK, Eilers RE, Neal AR, et al.: Precursors to speech in infancy: The prediction of speech and lan-guage disorders. J. Commun. Disord. 1999; 32(4): 223–245. PubMed Abstract \| Publisher Full Text\n\nPaul R: Profiles of toddlers with slow expressive language development. Topics in Disorders. 1991; 11(4): 1–13. Publisher Full Text\n\nPaul R, Jennings P: Phonological behavior in toddlers with slow expressive language development. J. Speech Lang. Hear. Res. 1992; 35(1): 99–107. PubMed Abstract \| Publisher Full Text\n\nPaul R, Murray C, Clancy K, et al.: Reading and metaphonological outcomes in late talkers. J. Speech Lang. Hear. Res. 1997; 40(5): 1037 1047.\n\nPreston JL, Frost SJ, Mencl WE, et al.: Early and late talkers: School-age language, literacy and neurolinguistic differences. Brain. 2010; 133(8): 2185–2195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReilly S, Wake M, Bavin EL, et al.: Predicting language at 2 years of age: a prospective community study. Pediatrics. 2007; 120(6): e1441–e1449. PubMed Abstract \| Publisher Full Text\n\nRescorla L: Language and reading outcomes to age 9 in late-talking toddlers. J. Speech Lang. Hear. Res. 2002; 45: 360–371. PubMed Abstract \| Publisher Full Text\n\nRescorla L: Age 13 language and reading outcomes in late-talking toddlers. J. Speech Lang. Hear. Res. 2005; 48: 459–472. PubMed Abstract \| Publisher Full Text\n\nRescorla L: Age 17 language and reading outcomes in late-talking toddlers: Support for a dimensional perspective on lan-guage delay. J. Speech Lang. Hear. Res. 2009; 52: 16–30. PubMed Abstract \| Publisher Full Text\n\nRescorla L, Dale PS: Late talkers: Language development, interventions, and outcomes. Paul H. Brookes Publishing Company; 2013.\n\nRescorla L, Lee EC: Handbook of early language impairment in children. Nature. 2000; 1: 1–38.\n\nRescorla L, Ratner NB: Phonetic profiles of toddlers with specific expressive language impairment (SLI-E). J. Speech Lang. Hear. Res. 1996; 39(1): 153–165. PubMed Abstract \| Publisher Full Text\n\nReznick JS, Goldfield BA: Rapid change in language acquisition during the second year: Naming explosion or knowing explosion. Soc. Res. Child Dev. 1989.\n\nRice ML, Taylor CL, Zubrick SR: Language outcomes of 7-year-old children with or without a history of late language emergence at 24 months. J. Speech Lang. Hear. Res. 2008; 51: 394–407. Publisher Full Text\n\nRipley B, Venables W, Ripley MB: Package ‘nnet’. R package version.2016; 7: 3–12.\n\nRoberts MY, Kaiser AP: Early intervention for toddlers with language delays: a randomized controlled trial. Pediatrics. 2015; 135(4): 686–693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRose SA, Feldman JF, Jankowski JJ, et al.: A cognitive cascade in infancy: Pathways from prematurity to later mental development. Intelligence. 2008; 36(4): 367–378. PubMed Abstract \| Publisher Full Text\n\nRowe ML: A longitudinal investigation of the role of quantity and quality of child directed speech in vocabulary develop-ment. Child Dev. 2012; 83: 1762–1774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowe ML, Snow CE: Analyzing input quality along three dimensions: Interactive, linguistic, and conceptual. J. Child Lang. 2020; 47(1): 5–21. PubMed Abstract \| Publisher Full Text\n\nScarborough HS, Dobrich W: Development of children with early language delay. J. Speech Lang. Hear. Res. 1990; 33(1): 70–83. Publisher Full Text\n\nSchjølberg S, Eadie P, Zachrisson HD, et al.: Predicting language development at age 18 months: data from the Norwegian Mother and Child Cohort Study. J. Dev. Behav. Pediatr. 2011; 32(5): 375–383. PubMed Abstract \| Publisher Full Text\n\nSilva PA: The prevalence, stability, and significance of developmental language delay in preschool children. Dev. Med. Child Neurol. 1980; 22(6): 768–777. PubMed Abstract \| Publisher Full Text\n\nSt Clair MC, Pickles A, Durkin K, et al.: A longitudinal study of behavioral, emotional and social diffi-culties in individuals with a history of specific language impairment (SLI). J. Commun. Disord. 2011; 44(2): 186 199.\n\nStanton-Chapman TL, Chapman DA, Bainbridge NL, et al.: Identification of early risk factors for language impairment. Res. Dev. Disabil. 2002; 23(6): 390–405. PubMed Abstract\n\nStern DN: The interpersonal world of the infant: A view from psychoanalysis and developmental psychology. Routledge; 2018.\n\nTenenbaum EJ, Sobel DM, Sheinkopf SJ, et al.: Attention to the mouth and gaze following in infancy predict language development. J. Child Lang. 2015; 42(6): 1173–1190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThal DJ, Marchman VA, Tomblin JB: Late-talking toddlers: Characterization and prediction of continued delay.2013.\n\nTomasello M, Kruger AC: Joint attention on actions: Acquiring verbs in ostensive and non-ostensive contexts. J. Child Lang. 1992; 19(2): 311–333. PubMed Abstract \| Publisher Full Text\n\nTomasello M: Joint attention as social cognition.Moore C, Dunham PJ, editors. Joint attention: Its origins and role in de-velopment. Hillsdale, NJ: La; 1995.\n\nVandekerckhove J, Matzke D, Wagenmakers EJ: Model comparison and the principle of parsimony. Oxford handbook of computational and mathematical psychology. 2015; pp. 300–319.\n\nVihman MM, Macken MA, Miller R, et al.: From babbling to speech: A re-assessment of the continuity issue. Language. 1985; 61: 397–445. Publisher Full Text\n\nWagenmakers EJ, Farrell S: AIC model selection using Akaike weights. Psychon. Bull. Rev. 2004; 11(1): 192–196. Publisher Full Text\n\nWallentin M: Gender differences in language are small but matter for disorders. Handb. Clin. Neurol. 2020; 175: 81–102. PubMed Abstract \| Publisher Full Text\n\nWasserstein RL, Schirm AL, Lazar NA: Moving to a world beyond “p< 0.05”. Am. Stat. 2019; 73(sup1): 1–19. Publisher Full Text\n\nWatt N, Wetherby A, Shumway S: Prelinguistic predictors of language outcome at 3 years of age. J. Speech Lang. Hear. Res. 2006; 49: 1224–1237. PubMed Abstract \| Publisher Full Text\n\nWhitehurst GJ, Fischel JE: Practitioner Review: Early Developmental language Delay: What if anything should the clinician do about it? J. Child Psychol. Psychiatry. 1994; 35(4): 613–648. PubMed Abstract \| Publisher Full Text\n\nWickham H: Data analysis. ggplot2: elegant graphics for data analysis.2016; 189–201.\n\nZambrana IM, Ystrom E, Pons F: Impact of gender, maternal education, and birth order on the development of lan-guage comprehension: a longitudinal study from 18 to 36 months of age. J. Dev. Behav. Pediatr. 2012; 33(2): 146 155.\n\nZambrana IM, Pons F, Eadie P, et al.: Trajectories of language delay from age 3 to 5: Persistence, recovery and late onset. Int. J. Lang. Commun. Disord. 2014; 49(3): 304–316. PubMed Abstract \| Publisher Full Text\n\nZubrick SR, Taylor CL, Rice ML, et al.: Late language emergence at 24 months: An epidemiological study of prevalence, predictors, and covariates. J. Speech Lang. Hear. Res. 2007; 50(6): 1562–1592. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "304096", "date": "09 Aug 2024", "name": "Mara Morelli", "expertise": [ "Reviewer Expertise Developmental psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments and Suggestions for Authors: Thank you for the opportunity to review this manuscript. This interesting and needed study investigated predictors of vocabulary size that may help professionals to identify children's performance as late, typical, or early talkers. A strength of this study is the age points included. While most of the research has included children starting at ten months of age, this study investigated predictors as early as three months. Minor edits are needed.\nParticipants: In the theoretical introduction, the authors mention the fact that gender can impact language acquisition. How can the imbalance between males and females be justified in your sample?\nParticipants: Why were only mothers chosen for the dyad study? Work on fathers exists. It would be important to specify this choice in the problematic section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "309659", "date": "12 Aug 2024", "name": "Maria Chiara Pino", "expertise": [ "Reviewer Expertise - Study of developmental trajectoriesof socio-emotional skills in children with typical and atypical development. -Linguistic and socio-cognitivedevelopment: relationship between cognitive and linguistic domains at different stages oflearning", "predictive indices of development. -Positive adaptation in the lifecycle: multidimensional analysis of life styles and choices and adaptation tothe environment", "both school and community", "in adolescents and young adults. -Application of technology-basedinterventions to enhance social and emotional skills in children with atypicaldevelopment. - Application of models and techniques characterising the study of socio-emotional skills. Studyof the relationship between reading and language development and the readingprocesses involved in the promotion of social-emotional", "affective andrelational skills." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLogriego et al.’s study represents an important contribution to research on the predictors of language development during the first two years of life. The subject matter is topical because the prevalence of late talkers and their difficulties in other domains might have increased due to the atypical constraints associated with the COVID-19 pandemic and widening economic and educational inequalities. The authors identify the interactive behaviours—between 3 and 18 months—that best predict early, typical, or late talkers (by the 24th month). The results show they can manifest as early as 3 months for vocal production, 6 months for facial gazing at the mother, and 12 months for gestural production.\n\nThis well-written and well-designed study complements the emergent literature on how social cues produced by a child from 3 months can predict subsequent language outcomes (i.e., the breadth of productive vocabulary at 2 years).\n\nThe theoretical discussion of the early markers (not only linguistic ones) and developmental trajectories of language is sound, although it would benefit from the inclusion of other papers on this topic (see below). A strong point of the study is the longitudinal design, which is based on data from 3, 6, 9, and 12-month-old infants.\nI would like to make the following suggestions:\n\nThe principal aim of the study should be specified before the method section in the Abstract. The authors should specify whether the descriptive values differ between groups in Table 1 (notwithstanding the small sample size). The following studies might be cited regarding the state of the art in the field:\n\nCaselli, M. C., Rinaldi, P., Stefanini, S., Volterra, V. Early action and gesture “vocabulary” and its relation with word comprehension and production. Child Dev. 2012, 83, 526–542. D’Amico, S., De Cagno, A. G., Levorato, M. C., Rossetto, T., Sansavini A. (2022). Il Disturbo Primario di\n\nLinguaggio. Oltre la Consensus Conference. Trento: Erickson, ISBN: 9788859025900. Sansavini, A., Bello, A., Guarini, A., Savini, S., Stefanini, S., Caselli, M. C. Early development of gestures, object-\n\nrelated-actions, word comprehension and word production and their relationships in Italian infants: A\n\nlongitudinal study. Gesture 2010, 10, 52–85. Sansavini, A., Favilla, M. E., Guasti, M. T., Marini. A., Millepiedi, S., Di Martino. M. V., Vecchi. S., Battajon. N.,\n\nBertolo, L., Capirci, O., Carretti, B., Colatei, M. P., Frioni, C., Marotta, L., Massa, S., Michelazzo, L.,\n\nPecini, C., Piazzalunga. S., Pieretti, M., Rinaldi, P., Salvadorini, R., Termine, C., Zuccarini, M., D’Amico. S.,\n\nDe Cagno, A. G., Levorato, M. C., Rossetto, T., Lorusso, M. L. Developmental Language Disorder: Early\n\nPredictors, Age for the Diagnosis, and Diagnostic Tools. A Scoping Review. Brain Sci. 2021 May\n\n17;11(5):654. doi: 10.3390/brainsci11050654. PMID:\n\n34067874; PMCID: PMC8156743.\n\nThe authors cite Rowe et al.’s (2020) important contribution to the field in their bibliography, but I cannot find a reference to it in the main text of the manuscript. They should consider their findings in light of Rowe et al.’s proposed classification.\n\nFinally: did the authors consider other factors that can affect language development, for example, a mother’s speech, preterm birth, nursery attendance, frequent otitis, and other relevant health conditions? These aspects can be seen as limits and possible future perspectives.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-798
https://f1000research.com/articles/12-855/v1	20 Jul 23	{ "type": "Study Protocol", "title": "Comparative evaluation and correlation of variations in articular disc morphology as assessed by automated segmentation using deep learning on magnetic resonance imaging (MRI) images in Class II (vertical) TMD cases, Class II (horizontal) TMD cases and Class I non-TMD cases", "authors": [ "Aathira Surendran", "Sunita Shrivastav", "Gaurav Srivastav", "Sunita Shrivastav", "Gaurav Srivastav" ], "abstract": "Introduction: Temporomandibular disorder (TMD) encompasses several clinical manifestations, which are characterized by temporomandibular joint and masticatory muscle discomfort and dysfunction (TMJ). The best imaging technique for evaluating TMJ is magnetic resonance imaging (MRI), which makes it possible to see the anatomical and pathological characteristics of every joint component. In recent years, convolutional neural networks -based deep learning algorithms have been favoured because of their outstanding capability in recognizing objects in medical images. The objective of this study is to assess, compare and co-relate articular disc morphology by automated segmentation using deep learning on MRI images in skeletal Class II (vertical growth pattern) TMD cases as compared to skeletal Class II (horizontal growth pattern) TMD cases and Class I non-TMD cases Methods: Grading of skeletal Class II (vertical growth pattern) cases and skeletal Class II (horizontal growth pattern) cases based on severity of TMD will be carried out using diagnostic criteria for temporomandibular disorders. Bilateral sagittal as well as coronal MRI images will be obtained. A convolutional neural network (CNN) encoder-decoder named U-Net will be used to segment the articular disc on MRI. Understanding the nature of variations between Class I and both types of Class IIs will help orthodontists to better predict the potential risk for the development of TMDs and accordingly take precautions while doing treatment in such cases. Moreover, it can be used to automate TMD diagnosis and other smart applications. Conclusions: This study will aid in identifying articular disc morphology on MRI. The deep learning algorithms with effective data augmentation may perform better in MRI readings than human clinicians when using the same data, which will be advantageous for TMD diagnosis.", "keywords": [ "temporomandibular disorder", "TMD", "articular disc", "Class II TMD cases", "automated segmentation", "deep neural network", "convolutional neural network" ], "content": "Introduction\n\nTemporomandibular disorder (TMD) encompasses several clinical manifestations which is characterized by temporomandibular joint and masticatory muscle discomfort and dysfunction (TMJ).1 TMD is frequently characterized by facial and pre-auricular regional pain, malocclusion, limited jaw movement, and clicking and locking of the TMJ.2\n\nThe expected incidence rate of TMD at first onset is 3.9%, with mild to severe pain and impairment, based on an American prospective cohort study of the general public. In industrialized nations, it affects 5–12% of the population.3 As reported previously in the literature, TMD is more common in cases with skeletal Class II (vertical growth pattern) followed by skeletal Class II (horizontal growth pattern) as compared to Class I cases. This was first reported by Pancherz in 1999.4\n\nThe best imaging technique for evaluating TMJ is magnetic resonance imaging (MRI), which makes it possible to see both the anatomical and pathological characteristics of every joint component. The shape and location of the articular disc, the mandibular condyle’s shape and surface characteristics, atypical bone marrow signal in the mandible and temporal bone can all be assessed using an MRI.5 Disc displacement or deformation, an intracapsular condition affecting the disc-condylar complex, is one of the most prominent subgroups in individuals with TMD who have articular abnormality, with an incidence of 30–60%. An MRI is necessary for assessing variations in articular disc morphology and to predict the treatment outcome.3\n\nArtificial intelligence (AI)-based dental applications have been researched to simplify dental care and enhance the health of more cases at a cheap cost and are attracting interest in a variety of clinical fields. By implementing AI-based dental applications, dental professionals can reduce the amount of time they spend performing regular tasks, which will enable them to provide more individualized, preventive, and collaborative dental care. In recent years, convolutional neural networks (CNN)-based deep learning algorithms have been more favoured because of their outstanding capabilities to recognize objects in medical images. Furthermore, as computational power has increased and open-source frameworks have become more common, CNN development has been dramatically facilitated. As a result, for detection and segmentation purposes, deep learning has been extensively used, showing encouraging results. There have been reports of a number of deep neural network topologies, including the CNN-derived fully convolutional network’s U-Net and SegNet variants.6\n\nClinicians and radiologists will be able to save time and interpret pictures more accurately if they can correctly identify the TMJ area’s major structures on MRI imaging. It can also be the basis for a lot of clever applications, including automatically diagnosing TMDs. It is without doubt important to accomplish this goal to automatically segregate TMJ structures from MRI images. Using these deep learning techniques, the mandibular condyle, articular eminence, and TMJ articular disc all are automatically detected. The TMJ segmentation of anatomical features in MRI volumes is made innovative in the current diagnostic image analysis investigation employing CNN-based DL methods.\n\n\n\n1. To evaluate articular disc morphology in Class I (Non-TMD) cases, skeletal Class II (vertical growth pattern) TMD cases, skeletal Class II (horizontal growth pattern) TMD cases.\n\n2. To compare articular disc morphology in skeletal Class II (vertical growth pattern) TMD cases with skeletal Class II (horizontal growth pattern) TMD cases and Class I (Non-TMD) cases.\n\n3. To correlate the variation in articular disc morphology with skeletal pattern.\n\n\nProtocol\n\nAn observational and analytical study will be conducted at Sharad Pawar Dental College, Datta Meghe Institute of Higher Education and Research (DMIHER), Sawangi, Wardha, Maharashtra in collaboration with the Faculty of Engineering and Technology (FEAT), DMIHER and the Department of Radiology, Acharya Vinobha Bhave Rural Hospital (AVBRH), Sawangi, Wardha.\n\nA total of 90 adult cases (Class I, Class II (vertical and horizontal growth pattern)) will be chosen from the Sharad Pawar Dental College’s outpatient department (OPD) of orthodontics and dentofacial orthopaedics in Sawangi, Wardha.\n\nInclusion criteria\n\n• Class I malocclusion cases.\n\n• Skeletal Class II (vertical growth pattern) and TMD cases.\n\n• Cases with skeletal Class II (horizontal growth pattern) and TMD.\n\n• Cases with permanent dentition.\n\n• Older than 18 years of age.\n\n• The cases will be classified into Class I and Class II (vertical and horizontal) by cephalometric measurements as shown in the Table 1.\n\nExclusion criteria\n\n• Skeletal Class II (vertical growth pattern) non-TMD cases.\n\n• Non-TMD Class II (horizontal growth pattern) cases.\n\n• Class III cases.\n\n• Cases with myofascial pain dysfunction (MPD) and myalgia.\n\n• Prior treatment involving temporomandibular joint surgery.\n\n• Cases with any skeletal disorders such as cherubism, osteoporosis.\n\n\nMethods\n\nThe patient will be diagnosed according to the diagnostic criteria for temporomandibular disorders (DC/TMD). Each participant will be asked for their written, informed consent. Based on the severity of TMD, skeletal Class II (vertical and horizontal growth pattern) cases will be graded using DC/TMD criteria. Both bilateral saggital and coronal MRI images will be obtained.\n\nThe articular disc of the TMJ on MRI will be identified and manually segmented with the help of technical expertise from the Faculty of Artificial Intelligence and Machine Learning (AIML) at FEAT College, Datta Meghe Institute of Higher Education and Research. After segmentation those images will be used as the dataset. The dataset showing the normal position of articular discs will be randomly split into a training data set and test set. To reduce the overfitting of the network, the dropout layer is placed behind the convolutional layers and max-pooling layers. Region of interests around the articular disc will be extracted from the datasets.\n\nRegion of interests will be automatically cropped from the images using Python 3.11 algorithms. Convolutional neural network (CNN) encoder-decoder named U-Net model architecture using the software Visual Studio Code with the Python programming language will be used to segment the articular disc on MRI. The variation in articular disc morphology will be evaluated and compared in Class II (vertical and horizontal growth pattern) with Class I Non-TMD cases using deep learning algorithms on MRI.\n\nPrimary outcome: To evaluate articular disc morphology in skeletal class II vertical and horizontal growth pattern.\n\nSecondary outcome: To compare and correlate the variation in articular disc morphology with skeletal pattern.\n\nTertiary outcome: For doctors and radiologists, the ability to precisely recognise important features in MRI of the TMJ region will save time and increase accuracy. Also understanding the nature of variations between Class I and both types of Class IIs will help orthodontists to better predict the potential risk for development of TMDs and accordingly take precautions while carrying out treatment in such cases. Moreover, it can be used to automate TMD diagnosis and other smart applications. This is why automated segmentation of TMJ structures with MRI is clearly necessary.\n\nBias will be minimized by random selection of patients based on the inclusion and exclusion criteria.\n\nThe calculation of sample size was carried out as follows:\n\nFormula using mean difference\n\nZα = 1.64\n\nα = Type I error at 5% at both sides two tailed\n\nΖβ = 0.84 (1 - β) = power at 80%\n\nσ = standard deviation\n\nPrimary variable: Articular disc variation (mm)\n\nClass I TMD group = 1.38 ± 0.20 (John et al. (2020)7)\n\nClass II (horizontal growth) TMD (mm)) group = 1.51 ± 0.20 (John et al. (2020)7)\n\nClinically relevant difference = 0.13\n\nPooled standard deviation = (0.20+0.20)/2 = 0.2\n\nTotal samples required = 30 per group.\n\nTotal sample size = 90\n\nThe sample would be divided into three groups based on the inclusion and exclusion criteria:\n\nGroup A (control group): 30 Class I (Non-TMD) cases.\n\nGroup B: 30 skeletal Class II (vertical growth pattern) cases.\n\nGroup C: 30 skeletal Class II (horizontal growth pattern) cases.\n\nAll the demographic outcome data will be presented using descriptive statistics for categorial variables in terms of frequency and percentage for continuous variables in terms of mean, standard deviation and median. Results will be analysed using SPSS version 27 (RRID:SCR_019096) for statistical analysis. Outcome variables will be tested for normality using the Kolmogorov-Smirnov test for continuous data.\n\nAll the samples will be distributed amongst the category of Class I non-TMD, Class II (vertical growth pattern) TMD cases and Class II (horizontal growth pattern) TMD cases as per the cephalometric measurements. Articular disc variation at different positions will be evaluated between these three groups using ANOVA or Kruskal Wallis test.\n\nANOVA or a Kruskal Wallis test will be used to find the result amongst the three groups for the outcome variable for the significance in mean difference. If the data are normally distributed an ANOVA test will be used and if the data are not normally distributed a non-parametric test (Kruskal Wallis) will be used to find the significant difference.\n\nThe original research will be published in a research article of an index journal. To ensure the original study receives the most exposure possible, the trial results will be released as open access.\n\nThe deep learning model is currently being prepared and the study will commence in May 2023.\n\n\nDiscussion\n\nNowadays, a lot of people have TMD due to the increased stress brought on by our fast-paced society. TMD is regarded as a collection of orofacial joint and muscle problems marked by pain, abnormal joint noises, and uneven jaw function. Although only 30% of subjects may be aware of such symptoms, the majority of research show that at least 50% of people have at least one symptom (such as muscle tenderness or joint clicking).8\n\nThere is variation in the morphology of the hard and soft tissue structures of TMJ specially of the articular disc. At times visualization and clarity of joint morphology and the articular disc may hinder the accuracy of diagnosis. What is the extent of variation and what type of disc morphology may cause a higher potential risk of TMD needs to be explored for early and more accurate prediction.\n\nHirata et al. (2007) studied the articular disc shape and location in patients with disc displacement, as well as the shape of the temporomandibular joint’s articular eminence. In the study, 14 individuals with bilateral disc displacement and no unilateral reduction were included. For evaluation, they employed magnetic resonance scans and showed that the chance of non-reducing disc displacement may be influenced by the shape of the articular disc and eminence.9\n\nIn 2020, John et al., observed that a Class II condition with a vertical growth pattern had the highest probability of developing a TMD with internal disk derangements and reduced anterior and posterior joint spaces compared to Class II with a horizontal growth pattern or Class I condition.7\n\nIto et al. (2022), conducted a study that assessed fully segmentation of the temporomandibular joint’s articular disc by automated means. Ten patients with anterior disc displacement and ten healthy control participants with normal articular discs were enrolled in the study. On MRI, they applied a semantic segmentation method based on deep learning. The study showed that this method for segmenting articular discs using automated deep learning on MRI generated encouraging preliminary results suggesting that the approach might be employed in clinical practice for the evaluation of temporomandibular disorders.3\n\nBased on the findings of these previous studies, this planned study would enable us to recognize changes in articular disc shape, changes in articular disc location, and morphological abnormalities that can be evaluated by the deep learning network. The outcome of the treatment and diagnostic efficiency for TMD’s may improve using this technique.\n\nEthical approval has been granted by the Institutional Review Board of Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha. (Reference number: DMIHER (DU)/IEC/2023/570 on 06/02/2023).", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: STROBE checklist for ‘Comparative evaluation and correlation of variations in articular disc morphology as assessed by automated segmentation using deep learning on magnetic resonance imaging (MRI) images in Class II (vertical) temporomandibular joint (TMD) cases, Class II (horizontal) TMD cases and Class I non-TMD cases’, https://www.doi.org/10.5281/zenodo.7853465.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\ndos Santos EA , Peinado BRR, Frazão DR, et al.: Association between temporomandibular disorders and anxiety: A systematic review. Front. Psychiatry. 2022 Oct 13; 13: 990430. Publisher Full Text\n\nScrivani SJ, Keith DA, Kaban LB: Temporomandibular Disorders. N. Engl. J. Med. 2008 Dec 18; 359(25): 2693–2705. Publisher Full Text\n\nIto S, Mine Y, Yoshimi Y, et al.: Automated segmentation of articular disc of the temporomandibular joint on magnetic resonance images using deep learning. Sci. Rep. 2022; 12(1): 221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuf S, Pancherz H: Temporomandibular joint remodeling in adolescents and young adults during Herbst treatment: A prospective longitudinal magnetic resonance imaging and cephalometric radiographic investigation. Am. J. Orthod. Dentofac. Orthop. Off. Publ. Am. Assoc. Orthod. Its Const. Soc. Am. Board Orthod. 1999 Jun; 115(6): 607–618. Publisher Full Text\n\nAiken A, Bouloux G, Hudgins P: MR Imaging of the Temporomandibular Joint. Magn. Reson. Imaging Clin. N. Am. 2012 Aug; 20(3): 397–412. Publisher Full Text\n\nLi M, Punithakumar K, Major PW, et al.: Temporomandibular joint segmentation in MRI images using deep learning. J. Dent. 2022; 127: 104345. PubMed Abstract \| Publisher Full Text\n\nJohn ZAS, Shrivastav SS, Kamble R, et al.: Three-dimensional comparative evaluation of articular disc position and other temporomandibular joint morphology in Class II horizontal and vertical cases with Class I malocclusion. Angle Orthod. 2020 Sep; 90(5): 707–714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen S, Ye M, Wu M, et al.: MRI and DC/TMD methods analyze the diagnostic accuracy of the change in articular disc of temporomandibular joint. Comput. Math. Methods Med. 2022; 2022: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHirata FH, Guimarães AS, de Oliveira JX , et al.: Evaluation of TMJ articular eminence morphology and disc patterns in patients with disc displacement in MRI. Braz. Oral Res. 2007 Sep; 21(3): 265–271. PubMed Abstract \| Publisher Full Text" }	[ { "id": "232583", "date": "12 Feb 2024", "name": "Martina Ferrillo", "expertise": [ "Reviewer Expertise Temporomandibular 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\nTemporomandibular disorders (TMD) are a collective of various symptoms caused by abnormalities in the temporomandibular joint (TMJ) and related structures. It affects 20% to 40% of the general population. Lifestyle habits, like alcohol consumption and smoking, are considered to be linked to TMD. This study aimed to assess, compare and co-relate articular disc morphology by automated segmentation using deep learning on MRI images in skeletal Class II (vertical growth pattern) TMD cases as compared to skeletal Class II (horizontal growth pattern) TMD cases and Class I non-TMD cases.\nThe study is of scientific interest and in line with the aims of the Journal. However, there are some issues that should be added.\nI suggest improving the abstract section:\n“temporomandibular joint and masticatory muscle discomfort and dysfunction (TMJ).” Please move “(TMJ)” after “temporomandibular joint”.\n\nI suggest improving the introduction section on TMD:\nClassification. Please refer to the Diagnostic Criteria for TMD (DC/TMD) Axis I. Thus, report that TMD could be divided in Group I: muscle disorders (including myofascial pain with and without mouth opening limitation); Group II: including disc displacement with or without reduction and mouth opening limitation; Group III: arthralgia, arthritis, and arthrosis.). (cite and refer to: Schiffman et al., 20141).\n\n“and pre-auricular regional pain, malocclusion, limited jaw movement, and clicking and locking of the TMJ “. Please report that TMD patients may present overlapping symptoms whit other chronic pain conditions, including headache, fibromylagia, and neurological conditions, probably through the phenomenon of central sensitization (mainly allodynia and hyperalgesia).\n\nPlease report more recent literature on conservative approaches and report new proposed treatment in the scientific literature (Agostini et al., 20232; Madani et al., 20203).\nMethods:\n“A total of 90 adult cases” - Please move this information in the Result Section.\n\nUse in all the text and tables “P” or “p-value”.\n\nClass II patients were assessed using ANB or Wits? Or Both? Please specify.\n\nThe same question for Class I.\n\nAdd reference for DC/TMD.\n\nResults are well described. Discussion should be improved reposting more recent literature on this topic.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11971", "date": "15 Jul 2024", "name": "Aathira Surendran", "role": "Author Response", "response": "Thank you ma'am for your thorough analysis of my article. I have made the following changes based on your review. 1. Classification. Please refer to the Diagnostic Criteria for TMD (DC/TMD) Axis I. Thus, report that TMD could be divided in Group I: muscle disorders (including myofascial pain with and without mouth opening limitation); Group II: including disc displacement with or without reduction and mouth opening limitation; Group III: arthralgia, arthritis, and arthrosis.). (cite and refer to: Schiffman et al., 20141). Response- i have included this in the introduction section. 2. “and pre-auricular regional pain, malocclusion, limited jaw movement, and clicking and locking of the TMJ “. Please report that TMD patients may present overlapping symptoms whit other chronic pain conditions, including headache, fibromylagia, and neurological conditions, probably through the phenomenon of central sensitization (mainly allodynia and hyperalgesia). Response- i have reported it. 3. Please report more recent literature on conservative approaches and report new proposed treatment in the scientific literature (Agostini et al., 20232; Madani et al., 20203). Response- I have added this literature in the introduction 4. Methods: “A total of 90 adult cases” - Please move this information in the Result Section Use in all the text and tables “P” or “p-value”. Class II patients were assessed using ANB or Wits? Or Both? Please specify. The same question for Class I. Add reference for DC/TMD Response- as this is a study protocol there isn't a result section so I couldn't move the information of total cases. I have made the corrections like specified the method used for assessing Class II and Class I patients and have added the reference for DC/TMD." } ] } ]	1	https://f1000research.com/articles/12-855
https://f1000research.com/articles/11-1574/v1	23 Dec 22	{ "type": "Systematic Review", "title": "Factors contributing to moral distress among intensive care nurses: A scoping review", "authors": [ "Amina Mussa Ahmad", "Wegdan Bani-Issa", "Fatma Refaat", "Wegdan Bani-Issa", "Fatma Refaat" ], "abstract": "Background: The intensive care unit (ICU) is a busy and complex workplace, and several work-related and personal factors are known to make ICU nurses more vulnerable to moral distress than other healthcare professionals. It is crucial to identify these factors to guide future studies and preventive strategies. This scoping review explores such factors to present current knowledge on the factors that trigger moral distress and to guide future research by reviewing studies to explore and summarize factors that trigger moral distress in ICU nurses.\n\nMethods: The PubMed, EBSCO, and CINAHL Plus databases were searched to identify potentially relevant studies published between 2011 to 2022. Inclusion criteria: peer-reviewed studies published in English that provided results regarding factors causes or correlated to moral distress in ICU nurses. After removing 63 duplicates, 371 papers were excluded after title and abstract screening, leaving 47 articles for full-text screening. A further 30 articles were excluded as their outcomes did not include factors that caused moral distress, or were not specific to ICU nurses, so 17 studies were eventually analysed using qualitative content analysis through an inductive approach. The findings of the articles were extracted and coded independently by two authors, and data were grouped and categorized. Results: The content categories of factors contributing to ICU nurses' moral distress were organized into themes and subthemes. Four major themes were identified: Powerlessness, end-of-life care, ineffective teamwork, and personal characteristics of ICU nurses.\n\nConclusions: This review highlights the factors that contribute to moral distress in critical care nurses, which are mainly attributable to the organizational climate and the nature of the ICU clinical environment. Descriptive and intervention studies (experimental or action research) must investigate causality between identified variables to inform management strategies to improve support for ICU nurses’ coping relative to moral distress.", "keywords": [ "End of life care", "ICU", "Moral distress", "Powerlessness", "Nurses", "Teamwork", "Scoping review" ], "content": "Introduction\n\nThe intensive care unit (ICU) is a complex, unpredictable, and inherently stressful environment in which patient health status, clinical situations, and basic dynamics of the course of care can all change rapidly (Johnson-Coyle et al., 2016). Factors such as nurse shortages (i.e., low nurse-to-patient ratios), patient populations with high acuity, significant technological advancement, inexperienced nurses with insufficient training, and poor healthcare systems make ICU nurses particularly vulnerable to moral distress in the discharge of their duties (Wenwen et al., 2018). Moral distress is experienced frequently by all healthcare professionals, including nurses (Chiafery et al., 2018; Wenwen et al., 2018). It is a phenomenon experienced when a healthcare professional knows the ethically appropriate course of action, but finds themselves unable to pursue that action, due to internal or other external constraints (Hamric et al., 2012). It occurs when conflict exits between knowledge of the ethically right course of action and institutional, departmental, interpersonal, or legal constraints, and the ability of healthcare providers to change the situation or take a course of action (Vincent et al., 2020). In particular, moral distress is linked with alleged violations of the core values and duties of the individual healthcare professional (Colville et al., 2019).\n\nIn ICUs it is typically difficult to predict the survival chances of critically ill patients, and nurses caring for such patients face far more morally distressing situations than those caring for patients with more stable conditions in other departments. ICU nurses face the twin demands of highly critical and complex biomedical service deliver alongside end-of-life issues such as sudden death, and they face moral dilemmas relating to aggressive and/or futile end-of-life interventions (Wenwen et al., 2018).\n\nIn such work environments, nurses run the risk of being involved in decisions that might be considered morally wrong (Babamohamadi et al., 2021), such as invasive interventions on patients with terminal illnesses, requesting pointless tests or examinations, inadequate and ineffective treatment by colleagues, a lack of organizational support, and absence of balance of power among healthcare professionals, all of which have been identified as causative factors in moral distress among nurses (Berhie et al., 2020; Emple et al., 2021; Heydari et al., 2018; Robaee et al., 2018). Other factors include giving life-supporting treatments that prolong the process of dying, and adhering to relatives’ directions to proceed with life-supporting medicines that may not be in the best interests of patients themselves (Wenwen et al., 2018). All of these factors lead to inability of healthcare team to reach consensus regarding to the plan of care for patients, resulting in team conflict and morally distressing situations (Vincent et al., 2020).\n\nFurthermore, the unpredictable progress of diseases and the fear of infection to self and family members are additional factors contributing to moral distress among ICU nurses, which was starkly revealed during the COVID-19 pandemic (Romero-García et al., 2022). In such demanding environments, burnout and low morale affects ICU nurses’ mental and physical well-being, as well as their relationships with friends and family, and patient care (Witter et al., 2020).\n\nThe consequences of moral distress in ICU nurses can include sensations of rage, dissatisfaction, powerlessness, and guilt, which are associated with discouragement, burnout, anxiety, depression, deteriorating morale and teamwork, diminishing quality of care (QoC), and difficulties associated with patient safety, all of which relate to emotional and psychological reactions correlated with moral distress (Romero-García et al., 2022). Negative personal symptoms reported by ICU nurses include nightmares, insomnia, palpitations, and neck pain (Bani Issa et al., 2021; Soleimani et al., 2019). If nurses’ moral distress remains unresolved, they may become emotionally exhausted, which consecutively reduces the QoC they deliver to patients, and increases their intension to leave the profession (McAndrew et al., 2018).\n\nAs a significant issue in healthcare environments, moral distress requires prompt and rapid actions because of the danger it poses to ICU nurses’ capacity to deliver competent and ethical care, the wellbeing of service users, and the QoC provided to patients (Almutairi et al., 2019), so healthcare organizations should recognize related factors and devise solutions to prevent and resolve moral distress (Saleh et al., 2019). Nurse mangers, administrators, and policy makers should implement evidence-based strategies to lower and eliminate nurse moral distress. A high-priority recommendation is for nursing leadership to provide rigorous supports such as structured debriefings, as well as a supportive environment for counselling services and staff education, with an emphasis on addressing moral distress and coping strategies (Dacar et al., 2019). Several interventional studies aimed at reducing moral distress provided insufficient evidence on the effectiveness of related interventions to reduce the level or frequency of moral distress (Dacar et al., 2019).\n\nDecades of research have shown that moral distress is high; however, there is little evidence of effective interventions (Altaker et al., 2018). Gaining insight into the moral distress that nurses face is the first step toward deciding actions that can provide guidance and nurse retention, which is fundamentally important for the sustainability of healthcare systems (Karakachian and Colbert, 2019). A preliminary search of various databases such as PubMed, EBSCO, MEDLINE, and CINAHL in addition to textbooks and the Cochrane Library, revealed that several studies have been conducted to identify factors that might contribute to moral distress in healthcare professionals working in ICUs, but relatively few have explored the factors that cause moral distress among ICU nurses per se.\n\nGiven the impact of moral distress among ICU nurses, patient safety, and QoC (Hou et al., 2021; Wenwen et al., 2018), it is crucial first to identify the factors that contribute to ICU nurses’ moral distress to guide healthcare organizations in developing resilience strategies to help ICU nurses cope with moral distress, and to pave the way for future research on practical interventions to address risk factors. Therefore, the aim of this scoping review is to present current knowledge on the factors that trigger moral distress and to guide future research by reviewing related studies to explore and summarize various factors that trigger moral distress in ICU nurses. The specific research questions that this scoping review seeks to address are:\n\n1. What are the general characteristics of studies on factors contributing or correlated to moral distress among ICU nurses?\n\n2. What are the factors contributing to moral distress among ICU nurses?\n\n\nMethods\n\nThis scoping review presents the direction of existing and future studies by analysing the characteristics and contents of related research using the methodological framework of Arksey and O’Malley (2005) to review the factors that lead to moral distress. This method was chosen as a way for mapping the literature in this area, examining the breadth, scope, and nature of research activity, and identifying gaps in existing knowledge (Harris et al., 2015). The Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist was used to report the present study (Ahmad et al., 2022).\n\nA comprehensive search was done in the academic research databases PubMed, EBSCO host, and CINAHL Plus to review articles published in the area under study. A recent systematic review on moral distress among healthcare professionals reported a steady increase in the volume of publications on the topic since 2010 (Lamiani et al., 2017). Therefore, the search in this study scope was limited to articles published in the period between January 2010 to July 2022, in order to be able to review the most recent and relevant available scientific evidence published in this field, and to have a comprehensive understanding of the area under study and identify gaps in recent literature.\n\nData were collected using the main keywords on moral distress, intensive care, and nurses.\n\nThe inclusion and exclusion criteria are presented in Table 1. The inclusion criteria restricted the scope to peer-reviewed studies that provided results regarding factors identified as being causative in or correlated with moral distress among ICU nurses, published in English. Studies that identified factors causing general psychological distress, collection of the constructs from target groups other than ICU nurses, studies on moral distress that did not include factors causing moral distress as outcome variables, and articles published in languages other than English were excluded.\n\nThis scoping review considered both experimental and quasi-experimental study designs, along with analytical observational, descriptive observational, descriptive cross-sectional, and qualitative studies, focused on qualitative data including (but not limited to) designs such as phenomenology, grounded theory, ethnography, and qualitative description. In addition, systematic reviews that met the inclusion criteria were also considered.\n\nThe search results were imported into EndNote X9 and were de-duplicated based on their titles, and the automatically identified duplicates were additionally double-checked manually. Two reviewers screened the results independently against the eligibility criteria. Discrepancies were resolved through discussions or asking a third reviewer.\n\nTwo reviewers also investigated the full texts of relevant search results against the same criteria (above) involving a third reviewer in cases of disagreement. At the full-text screening stage, the reference lists of the relevant studies were also analysed to identify potential additional relevant studies not gleaned from the database searching process. Since one study may be associated with multiple reports or publications, we kept a record and cited all the reports of a single study to provide a better overview of the new research evidence.\n\nThe initial search resulted in a total of 1,005 articles. After checking their titles and abstracts, 117 were excluded because the title of the study did not mention moral distress, 152 were excluded after reading the title (due to identifying non-eligibility factors), and 47 articles did not meet the eligibility criteria after reading their abstracts. Additionally, 618 articles were duplicates (and were removed), thus the remaining 71 articles were reviewed in full-text versions. This resulted in the exclusion of 54 articles due to their outcomes not including factors that caused moral distress or considering outcomes not specific to ICU nurses (e.g., those concerned with healthcare professionals in general or other specialties), thus 17 studies were eventually analysed (see Figure 1).\n\nA data extraction form was created prior to data extraction to aid with the process (Ahmad et al., 2022). The form included data about the year of publication, the objectives, the country, the study design, the author(s), the participants, the sample size, the tool used in the study, and the study's findings. They were then divided into two categories for analysis, as follows: (1) general characteristics of the studies; and (2) outcome variables and findings, which demonstrated the main factors that contribute to moral distress among ICU nurses.\n\nThe general characteristics of the studies included country, measurement instrument, and study design, and results of the studies on factors contributing to moral distress were summarized, and the extracted data were documented with Microsoft Excel Version 2209 (Table 2).\n\nTwo reviewers read the included articles independently and repeatedly to gain an overall understanding of the factors contributing to moral distress. Each reviewer then independently constructed codes from the surface meaning of the text and collated it. The reviewers identified codes and themes from raw data using an inductive approach to organize the data, to reach consensus, a third reviewer resolved disagreements.\n\n\nResults\n\nOf the 17 studies, four were conducted in the US (23.5%), three in Italy (17.6%), three in Iran (17.6%), two in Canada (11.8%), one each in South Korea (5.9%), Sweden (5.9%), Thailand (5.9%), Turkey (5.9%), and the UK (5.9%), and one was a review study (Ahmad et al., 2022).\n\nRegarding study design, 10 studies used a descriptive correlational design (58.8%), five used a qualitative design (29%), one used mixed-methods (5.8%), and one used an analytical review design (5.8%). Finally, regarding the measurement tools used to identify factors causing moral distress in critical care nurses, five quantitative studies used the 21-item Moral Distress Scale-Revised (29.4%), two used the 21-item Moral Distress Scale Neonatal-Pediatric Version (11.8%), two used the 32-item Moral Distress Scale (11.8%), and another study used the Jameton’s Moral Distress Questionnaire (5.8%). The mixed-methods design study used the Measure of Moral Distress for Healthcare Professionals (MMD-HP) (5.8%), whereas qualitative outcomes used semi-structured interviews in five studies (29.4%) and focus groups in one study (5.8%) (Table 3).\n\nTwo reviewers grouped and categorized data from the primary independent coding of the articles; data were derived and grouped; disagreements were resolved through discussion in online meetings between the reviewers by the third reviewer; and the groups were organized into themes and subthemes. Four major themes of factors associated with moral distress in ICU nurses were identified: powerlessness, end-of-life care, ineffective teamwork, and personal characteristics of ICU nurses (Table 4).\n\n\nDiscussion\n\nThe intensive care unit is a chaotic and stressful setting in which nurses are constantly confronted with disruptive factors that expose them to moral distress. This is an overwhelming environment of increasing daily admissions, transfer, and deaths; these particular stressors exacerbate underlying weaknesses common in healthcare systems worldwide, such as low nurse-patient ratios, overwork, burnout, and poor life-work balance etc., all of which are independently related with an increase in the absolute value of the moral distress of ICU nurses (Sannino et al., 2019).\n\nFrom the results of the present scoping review, it is clear that there are several factors that can lead to moral distress in critical care nurses, all of which negatively affect their personal and professional lives, and consequently the QoC they deliver, resulting in poorer patient outcomes (including clinical prognosis and wellbeing), service user satisfaction, and health system efficiency.\n\nAlmost all of the articles in this review indicated that the powerlessness of ICU nurses in the face of organizational obstacles and constraints is one of the main causes of their moral distress while working with patients (Altaker et al., 2018; Andersson et al., 2022; Browning, 2013; Choe et al., 2015; Epstein et al., 2019; McAndrew et al., 2018; Morley et al., 2022; Prompahakul et al., 2021; Sannino et al., 2019; Shoorideh et al., 2012).\n\nThe most important factor contributing to differences in levels of moral stress was the prevailing ethical climate, with the creation of a positive ethical climate in the organization being associated with lower levels of moral stress (Altaker et al., 2018). ICU nurses described discomfort when work-related tasks interfered with their ability to advocate for patients (i.e., to provide holistic nursing care), or when the economic benefit of the hospital was seen as taking precedence over human life, and in these contexts the ethical climate in the organization correlated negatively with moral distress (McAndrew et al., 2018). Similarly, Choe et al., (2015) revealed that administrative actions deemed to be ethically flawed often caused moral distress among participants, especially when hospitals’ financial objectives took precedence over respect for human life and rights (e.g., when patients were forced to be discharged or transferred regardless of their condition).\n\nRelated to their perceived powerlessness (Prompahakul et al., 2021), emphasized that nurses experienced moral distress within strong hierarchical organizations, wherein they lacked organizational support, which is one of the most important indicators of nursing work satisfaction. The power imbalance between nurses and medical healthcare professionals was related to hierarchical structures favouring the latter by McAndrew et al. (2018), who emphasized that medical values predominate over nursing values in the organizations in which they work, inhibiting nurses from executing their holistic role, and impeding the development of advanced nursing practice and nursing advocacy for patients in care decisions. The decision-making hierarchy often encourages physicians to ignore and disregard nurses’ opinions regarding patient care, contributing to the nurses’ moral distress (Morley et al., 2022; Shoorideh et al., 2012).\n\nFrom the results of this review, it was found that there was a strong hierarchical relationship between healthcare providers in the facilities where the studies were conducted. ICU nurses who are morally challenged felt powerless and helpless to stop treatment that they believed was ethically wrong, or where the care delivered did not meet required standards (McAndrew et al., 2018; Morley et al., 2022; Prompahakul et al., 2021). On this basis, we can emphasize the importance of a culture that promotes nurse autonomy and collaboration in decision-making related to patient care in different healthcare settings. Nurses perceived themselves as having little authority to make treatment decisions within the healthcare team. In many situations, nurses knew what was best for patients, but were unable to act due to a lack of practice and independence (Prompahakul et al., 2021). Concerns have been raised about the negative relationship between moral distress and autonomy and collaboration. Poor communication and collaboration can have serious consequences for patients and families if nurses do not feel valued in their professional interactions (McAndrew et al., 2018).\n\nInadequate resources, such as medical supplies, accessible beds, and staff, were another source of moral distress at the organizational level, as they undermined nurses’ ability to provide the best possible care to patients (Henrich et al., 2016; Prompahakul et al., 2021). Specific safety risks were reported, such as one case in which management pressed nurses to care for three patients at once, while also providing break relief for a fourth patient, which the nurses did not believe was safe (Henrich et al., 2016).\n\nNurse empowerment and organizational support appear to be important factors in reducing moral distress among ICU nurses (Altaker et al., 2018; Browning, 2013; Henrich et al., 2016; McAndrew et al., 2018; Morley et al., 2022; Prompahakul et al., 2021; Shoorideh et al., 2012). A study conducted to examine the relationship between moral distress and psychological empowerment among critical care nurses found that the total psychological empowerment score was negatively correlated with the frequency of moral distress, and multiple regression analysis revealed that empowerment was a significant predictor of the frequency of moral distress (Browning, 2013).\n\nEmpowerment of the individual nurse has been the focus of numerous educational interventions, particularly in the area of end-of-life care and team building, but has not resulted in a reduction in moral distress levels (Altaker et al., 2018), implying that more knowledge and perceptions of empowerment are not sufficient to reduce the moral burden of ICU nurses; actual implementation of nurse empowerment in the organization is required to reduce ICU nurse moral distress, although a roadmap to facilitate this remains lacking.\n\nThe powerlessness of ICU nurses described above was not limited to their interactions with other healthcare professionals and organizational administrators but extended to their exchanges with service users. Several studies indicated that inappropriate and unnecessary treatments directed by patients’ family members were among the main causes of moral distress among nurses (Browning, 2013; Henrich et al., 2016; McAndrew et al., 2018; Prompahakul et al., 2021). Surrogates are influential decision makers in some countries when patients lack decision-making capacity, which is relatively common in ICU, and healthcare professionals can be subject to legal action if they do not adhere to surrogates’ wishes, even if they perceive these to be contrary to the interests of the patients themselves. As a result, some participants expressed moral concerns when they knew that the decision chosen was inappropriate for a patient, but they had no choice but to follow the surrogate’s decision because of the latter’s power within the care decision-making process (Prompahakul et al., 2021).\n\nThis may be related to prosaic issues related to the amenities offered to service users by critical care facilities. Families often have an interest in maximizing patients’ stay and treatment in ICU, where healthcare professionals undertake all care activities (including for activities of daily living), and which are usually under the umbrella of ICU medical costs covered by insurance schemes, thus families may ignore options to transfer patients to other units or even to discharge them from hospital when the nurse may consider this to be the best option for the patient.\n\nIn other studies, it was found that some patients’ family members refused treatment for financial reasons, or because of the burden of caring for a patient, even if the patient had a high chance of recovery. In such cases, physicians often agreed to discontinue treatment without first attempting to convince the patients’ families, which angered nurses because they had no control over the situation (Choe et al., 2015; Prompahakul et al., 2021).\n\nThe reviewed studies concurred that moral distress was common among ICU nurses during the end-of-life decision-making process (Browning, 2013; Forozeiya et al., 2019; Henrich et al., 2016; Prompahakul et al., 2021; Sannino et al., 2015, 2019). Almost 80% of the morally distressing situations mentioned by participants in Prompahakul et al.,'s (2021) study were end-of-life situations, including unnecessary medical treatments, futile actions, and giving false hope to patients and families at the end of life. According to Henrich et al. (2016), nurses were concerned when aggressive care continued, even when the decision to switch to comfort care was made, when the decision was made on a weekend or at night; physicians commonly wanted to wait until after the weekend or until the next morning to implement the plan, for their own convenience rather than patients’ best interests. This finding was confirmed by Prompahakul et al. (2021), who found that ICU nurses were disturbed when they saw patients suffering as a result of overly aggressive and unnecessary treatments that they believed were not in the best interest of the patient\n\nFutile care has been described in several studies as a factor that triggers moral distress among ICU nurses (Andersson et al., 2022; Asayesh et al., 2018; Hiler et al., 2018; Karagozoglu et al., 2017; Prompahakul et al., 2021; Shoorideh et al., 2012). Futile care can be defined on the basis of survival or subsequent quality of life, and includes aggressive treatments or end-of-life interventions in patients with very low life expectancy or chance of recovery (Asayesh et al., 2018). According to Shoorideh et al. (2012), ICU nurses suffer moral distress due to unnecessary testing, unnecessary and expensive medications for patients nearing the end of life, unnecessary counselling, and cardiopulmonary resuscitation (Shoorideh et al., 2012). In critical care, the failure to set limits on futile treatments may exacerbate the experience of moral suffering in nurses (McAndrew et al., 2018)\n\nDeceptive information and false hope for patients and relatives were cited in several studies as a distressing act that caused moral distress for ICU nurses (Andersson et al., 2022; Browning, 2013; Forozeiya et al., 2019; Morley et al., 2022; Prompahakul et al., 2021). Nurses’ distress over withholding information from patients’ families was compounded by a stark discrepancy between what they saw physicians say directly to families and what they then said in private (Forozeiya et al., 2019), affirming a point highlighted by Choe et al., (2015). This information-framing resulted in families not receiving a complete picture of their patients’ deteriorating condition, leading to prolonged and aggressive care for dying patients. End-of-life care planning, including how physicians plan for end-of-life care and the extent of family involvement in end-of-life care decisions, was another aspect identified in two studies as contributing to moral distress (Forozeiya et al., 2019; Henrich et al., 2016).\n\nRelated to the hierarchical powerlessness described previously, poor communication and lack of cooperation with other healthcare professionals (particularly physicians) in caring for critically ill patients was a major cause of moral distress among ICU nurses in the articles reviewed (Choe et al., 2015; Henrich et al., 2016; Karagozoglu et al., 2017; Karanikola et al., 2014; McAndrew et al., 2018; Prompahakul et al., 2021; Shoorideh et al., 2012). When nurses reported higher levels of moral distress, they cited communication with the physician as a cause of medication errors (McAndrew et al., 2018). Similarly, Henrich et al. (2016) emphasized that ineffective physician communication with families was identified by nurses and other health professionals as a source of distress.\n\nDistress from not being heard during inter-professional conversations about end-of-life care typically magnified ICU nurses’ sense of powerlessness, despair, and frustration (McAndrew et al., 2018). Many studies highlighted many inappropriate peer behaviours that increased feelings of moral distress (McAndrew et al., 2018; Morley et al., 2022; Prompahakul et al., 2021). Neglecting patients and treating them unequally due to social hierarchy are unethical and morally questionable behaviours that triggered moral distress when encountered (Prompahakul et al., 2021).\n\nWorking with incompetent healthcare providers was another problem addressed in several studies (Andersson et al., 2022; Browning, 2013; Henrich et al., 2016; Karagozoglu et al., 2017; McAndrew et al., 2018; Prompahakul et al., 2021; Sannino et al., 2019). The nurses in the study conducted by Prompahakul et al., (2021) noted that their colleague’s incompetence threatened the patient’s integrity due to delayed treatment or inappropriate pain management. In another study, supporting a physician who provides incompetent care was found to be one of the main causes of the frequency and intensity of moral distress experienced by ICU nurses (McAndrew et al., 2018).\n\nIn two studies, conflicting care plans of the attending physicians for critically ill patients were cited as a cause of moral distress (Henrich et al., 2016; Shoorideh et al., 2012). Henrich et al. (2016) highlighted that the constant change in the care plan each week as new attending physicians took responsibility for patients was very stressful, as nurses had to explain to families why the direction of care had changed, especially if they disagreed with the change.\n\nAccording to recent data, formal communication processes that involve the entire team in departmental ethics discussions and nursing rounds, or that provide ethics or morale-boosting counselling services, can reduce factors contributing to moral distress (Altaker et al., 2018, p. 300).\n\nBased on the findings of the current review, the personal characteristics of nurses may play an important role in moral distress. Hiler et al. (2018) concluded that the age of ICU nurses was a statistically significant predictor of the severity of moral distress experienced (i.e., age was negatively correlated with moral distress). Similar findings were highlighted by Sannino et al. (2019), as they found a negative moderate correlation between ICU nurses’ age and level of moral distress. However, Browning (2013) reached the opposite conclusion, finding a positive relationship between the age of ICU nurses and their moral distress, emphasizing that the intensity of moral distress increased with the age of the nurse.\n\nNurses with a higher educational level were found to experience higher intensity of moral distress than those with relatively less education in a study by Altaker et al. (2018, p. 299), while Shoorideh and Ashktorab (2015) found no significant correlation.\n\nA negative correlation was found between duration of caregiving experience and level of moral distress by Patrizio Sannino et al. (2019, p. 5), contrary to the results of other studies which reported a positive correlation between work experience and the level of moral distress (Asayesh et al., 2018; Shoorideh & Ashktorab, 2015).\n\nMcAndrew et al. (2018) found that female nurses reported more moral distress than their male counterparts, while Shoorideh and Ashktorab (2015) found no significant correlation between gender and moral distress. Conversely, Sannino et al. (2015) reported that male nurses experienced a higher level of moral distress in a significantly higher percentage of cases than female nurses.\n\nThe literature included comprised full-text and research studies, so the content available in philosophical treatises, editorials, and dissertations could have extended the findings reported in this article.\n\nBecause the focus of the study was mainly on critical care settings, any generalizations of the findings are applicable only to this area of practice. However, by including neonatal and paediatric critical care nurses’ experiences in this study, the findings could be generalized to a broader range of critical care nurses.\n\n\nConclusion\n\nThis review has highlighted the factors that contribute to moral distress in critical care nurses. Many of the associated factors are due to the organizational climate and the nature of the ICU clinical environment. It is suggested that further studies should be conducted to examine the effects of some variables on moral distress, such as nurse autonomy, job satisfaction, length of duty hours, and nurses’ personal characteristics, which might exacerbate or cause moral distress.\n\nTherefore, descriptive and intervention studies, including both experimental and action research, are needed to investigate the causal relationship between the identified variables, and to determine the best strategy to help ICU nurses cope with and reduce the intensity of moral distress. Comparative studies are required to better understand the actual factors associated with moral distress, such as comparing the intensity of moral distress and contributing factors with a Magnet hospital, to examine the impact of organizational factors designed to improve nursing satisfaction and retention on moral distress.", "appendix": "Data availability\n\nFigshare: Factors Contributing to Moral Distress Among Intensive Care Nurses: A Scoping Review. https://doi.org/10.6084/m9.figshare.21422514.v1. (Ahmad et al., 2022).\n\nThe project contains the following underlying data:\n\n• File 1- PubMed search strategy performed July 13, 2022.jpg. (Search strategy used in this study).\n\n• Figure 1-PRISMA 2020 flow diagram.jpg. (Flowchart of study).\n\n• Table 1 – Eligibility criteria.jpg. (Inclusion criteria for studies).\n\n• Table 2 – Summary of included articles.docx. (List of final 17 articles in results).\n\n• Table 3 – General characteristics of included studies.jpg. (Characteristics of 17 final studies).\n\n• Table 4 – Content analysis.jpg. (Content analysis of final 17 studies).\n\nFigshare: Factors Contributing to Moral Distress Among Intensive Care Nurses: A Scoping Review. https://doi.org/10.6084/m9.figshare.21422514.v1. (Ahmad et al., 2022).\n\nThis project contains the following extended data:\n\n• Blank example of data extraction form.xlsx (blank extraction form used in this study).\n\nZenodo: PRISMA-Scr checklist for ‘Factors Contributing to Moral Distress Among Intensive Care Nurses: A Scoping Review’. https://doi.org/10.6084/m9.figshare.21422514.v1. (Ahmad et al., 2022).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAlmutairi AF, Salam M, Adlan AA, et al.: Prevalence of severe moral distress among healthcare providers in Saudi Arabia. Psychol. Res. Behav. Manag. 2019; 12: 107–115. PubMed Abstract \| Publisher Full Text\n\nAltaker KW, Howie-esquivel J, Cataldo JK: Relationships Among Palliative Care, Ethical Climate, Empowerment, and Moral Distress in Intensive Care Unit Nurses.2018; 27(4): 295–302.\n\nAhmad A, Bani-Issa W, Refaat F:Factors Contributing to Moral Distress Among Intensive Care Nurses: A Scoping Review. Dataset.2022. Publisher Full Text\n\nAndersson M, Nordin A, Engström Å: Critical care nurses’ perception of moral distress in intensive care during the COVID-19 pandemic – A pilot study. Intensive Crit. Care Nurs. 2022; 72: 103279. PubMed Abstract \| Publisher Full Text\n\nArksey H, O’Malley L: Scoping studies: towards a methodological framework. Int. J. Soc. Res. Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nAsayesh H, Mosavi M, Abdi M, et al.: The relationship between futile care perception and moral distress among intensive care unit nurses. J. Med. Ethics Hist. Med. 2018; 1–6.\n\nBabamohamadi H, Bakuei S, Paknazar F: Moral distress and its contributing factors among emergency department nurses: A cross-sectional study in Iran. Int. Emerg. Nurs. 2021; 56(October 2020): 100982. PubMed Abstract \| Publisher Full Text\n\nBani Issa W, Al Nusair H, AlTamimi A, et al.: Posttraumatic stress disorders and influencing factors during the COVID-19 pandemic: A cross-sectional study of frontline nurses. Int. Nurs. Rev. 2021; 69: 285–293. October. PubMed Abstract \| Publisher Full Text\n\nBerhie AY, Tezera ZB, Azagew AW: Moral distress and its associated factors among nurses in northwest amhara regional state referral hospitals, northwest ethiopia. Psychol. Res. Behav. Manag. 2020; 13: 161–167. PubMed Abstract \| Publisher Full Text\n\nBrowning AM: CNE article: moral distress and psychological empowerment in critical care nurses caring for adults at end of life. Am. J. Crit. Care. 2013; 22(2): 143–151. PubMed Abstract \| Publisher Full Text\n\nChiafery MC, Hopkins P, Norton SA, et al.: Nursing Ethics Huddles to Decrease Moral Distress among Nurses in the Intensive Care Unit. J. Clin. Ethics. 2018; 29(3): 217–226. PubMed Abstract\n\nChoe K, Kang Y, Park Y: Moral distress in critical care nurses: A phenomenological study. J. Adv. Nurs. 2015; 71(7): 1684–1693. PubMed Abstract \| Publisher Full Text\n\nColville GA, Dawson D, Rabinthiran S, et al.: A survey of moral distress in staff working in intensive care in the UK. J. Intensive Care Soc. 2019; 20(3): 196–203. PubMed Abstract \| Publisher Full Text\n\nDacar SL, Covell CL, Papathanassoglou E: Addressing Moral Distress in Critical Care Nurses: A Systemized Literature Review of Intervention Studies. Connect: The World of Critical Care Nursing. 2019; 13(2): 71–89. Publisher Full Text\n\nEmple A, Fonseca L, Nakagawa S, et al.: Moral Distress in Clinicians Caring for Critically Ill Patients Who Require Mechanical Circulatory Support. Am. J. Crit. Care. 2021; 30(5): 356–362. Publisher Full Text\n\nEpstein EG, Whitehead PB, Prompahakul C, et al.: Enhancing Understanding of Moral Distress: The Measure of Moral Distress for Health Care Professionals Enhancing Understanding of Moral Distress: The Measure of Moral Distress for Health Care Professionals. AJOB Empirical Bioethics. 2019; 1–12. Publisher Full Text\n\nForozeiya D, Vanderspank-Wright B, Bourbonnais FF, et al.: Coping with moral distress – The experiences of intensive care nurses: An interpretive descriptive study. Intensive Crit. Care Nurs. 2019; 53: 23–29. PubMed Abstract \| Publisher Full Text\n\nHamric AB, Borchers CT, Epstein EG: Development and Testing of an Instrument to Measure Moral Distress in Healthcare Professionals. AJOB Prim. Res. 2012; 3(December 2014): 1–9. Publisher Full Text\n\nHarris R, Sims S, Parr J, et al.: Impact of 12h shift patterns in nursing: A scoping review. Int. J. Nurs. Stud. 2015; 52(2): 605–634. PubMed Abstract \| Publisher Full Text\n\nHenrich NJ, Dodek PM, Alden L, et al.: Causes of moral distress in the intensive care unit: A qualitative study. J. Crit. Care. 2016; 35: 57–62. PubMed Abstract \| Publisher Full Text\n\nHeydari A, Ahrari S, Toghian Chaharsoughi N: Moral Distress in Nursing and Its Contributors in the Context of Iran. Health spiritual. Med. Ethics. 2018; 5(3): 44–50. Publisher Full Text\n\nHiler CA, Hickman RL, Reimer AP, et al.: Predictors of Moral Distress in a US Sample of Critical Care Nurses. Am. J. Crit. Care. 2018; 27(1): 59–66. PubMed Abstract \| Publisher Full Text\n\nHou Y, Timmins F, Zhou Q, et al.: A cross-sectional exploration of emergency department nurses’ moral distress, ethical climate and nursing practice environment. Int. Emerg. Nurs. 2021; 55(April 2020): 100972. PubMed Abstract \| Publisher Full Text\n\nJohnson-Coyle L, Opgenorth D, Bellows M, et al.: Moral distress and burnout among cardiovascular surgery intensive care unit healthcare professionals: A prospective cross-sectional survey. Can. J. Crit. Care Nurs. 2016; 27(4): 27–36.\n\nKaragozoglu S, Yildirim G, Ozden D, et al.: Moral distress in Turkish intensive care nurses. Nurs. Ethics. 2017; 24(2): 209–224. Publisher Full Text\n\nKarakachian A, Colbert A: Nurses’ Moral Distress, Burnout, and Intentions to Leave: An Integrative Review. J. Forensic Nurs. 2019; 15(3): 133–142. PubMed Abstract \| Publisher Full Text\n\nKaranikola MNK, Albarran JW, Drigo E, et al.: Moral distress, autonomy and nurse – physician collaboration among intensive care unit nurses in Italy. J. Nurs. Manag. 2014; 22(November 2012): 472–484. PubMed Abstract \| Publisher Full Text\n\nLamiani G, Borghi L, Argentero P: When healthcare professionals cannot do the right thing: A systematic review of moral distress and its correlates. J. Health Psychol. 2017; 22(1): 51–67. PubMed Abstract \| Publisher Full Text\n\nMcAndrew NS, Leske J, Schroeter K: Moral distress in critical care nursing: The state of the science. Nurs. Ethics. 2018; 25(5): 552–570. Publisher Full Text\n\nMorley G, Bradbury-Jones C, Ives J: The moral distress model: An empirically informed guide for moral distress interventions. J. Clin. Nurs. 2022; 31(9-10): 1309–1326. Publisher Full Text\n\nPrompahakul C, Keim-malpass J, Lebaron V, et al.: Moral distress among nurses: A mixed-methods study.2021. Publisher Full Text\n\nRobaee N, Atashzadeh-shoorideh F, Ashktorab T, et al.: Perceived organizational support and moral distress among nurses.2018; 1–7. Publisher Full Text\n\nRomero-García M, Delgado-Hito P, Gálvez-Herrer M, et al.: Moral distress, emotional impact and coping in intensive care unit staff during the outbreak of COVID-19. Intensive Crit. Care Nurs. 2022; 70(September 2021): 103206. PubMed Abstract \| Publisher Full Text\n\nSaleh ZN, Loghmani L, Rasouli M, et al.: Moral distress and compassion fatigue in nurses of neonatal intensive care unit. Electron. J. Gen. Med. 2019; 16(2): 2–5. Publisher Full Text\n\nSannino P, Giannì ML, Carini M, et al.: Moral Distress in the Pediatric Intensive Care Unit: An Italian Study. Front. Pediatr. 2019; 7(August): 1–7. PubMed Abstract \| Publisher Full Text\n\nSannino P, Giannì ML, Re LG, et al.: Moral distress in the neonatal intensive care unit: An Italian study. J. Perinatol. 2015; 35(3): 214–217. PubMed Abstract \| Publisher Full Text\n\nShoorideh FA, Ashktorab T: Relationship between ICU nurses’ moral distress with burnout and anticipated turnover. Nurs. Ethics. 2015; 22(1): 64–76. Publisher Full Text\n\nShoorideh FA, Ashktorab T, Yaghmaei F: Iranian intensive care unit nurses’ moral distress: A content analysis. Nurs. Ethics. 2012; 19(4): 464–478. PubMed Abstract \| Publisher Full Text\n\nSoleimani MA, Panarello B, Thin M, et al.: Spiritual well-being and moral distress among Iranian nurses. Nurs. Ethics. 2019; 26(4): 1101–1113. Publisher Full Text\n\nVincent H, Jones DJ, Engebretson J: Moral distress perspectives among interprofessional intensive care unit team members. Nurs. Ethics. 2020; 27(6): 1450–1460. PubMed Abstract \| Publisher Full Text\n\nWenwen Z, Xiaoyan W, Yufang Z: Moral distress and its influencing factors: A cross-sectional study in China. Nurs. Ethics. 2018; 25(4): 470–480. Publisher Full Text\n\nWitter T, Comber S, Hancock J, et al.: Understanding burnout and moral distress to build resilience: a qualitative study of an interprofessional intensive care unit team. Comprendre l’épuisement professionnel et la détresse morale afin de développer la résilience: une étude qualita. Can. J. Anesth./J. Can. Anesth. 2020; 67(11): 1541–1548. Publisher Full Text" }	[ { "id": "158631", "date": "20 Feb 2023", "name": "Intima Alrimawi", "expertise": [ "Reviewer Expertise My research focuses on improving the quality of care for vulnerable families and children with complex health concerns or critical illness and developing health services mainly in low and middle-income countries", "as well as developing the quality of nursing 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 scoping review explores the factors that would trigger moral distress among intensive care nurses by utilizing the scoping review approach. This is a very important topic that was not covered in other scoping reviews.\n\nIn the methodology section, the researchers thoroughly discussed the scientific approach that they followed to conduct this review.\n\nThe result section needs to be developed further, further discussion is needed around the selected studies and the final themes that did emerge from them.\n\nThe discussion section needs to take the themes to the next level and discuss the potential implication of each point. For example, if you think ineffective team function was a main trigger for moral distress, then what is the implication of this in the current practice, and how can we overcome this on the ground?\n\nThe authors can also add a recommendation section.\nThis scoping review explores the factors that would trigger moral distress among intensive care nurses through utilizing the scoping review approach and found that powerlessness, end-of-life care, ineffective teamwork, and the personal characteristics of ICU nurses were the main influencing factors of moral distress. The main strength of this article is that it discusses a main subject that would affect the nurses' practices by utilizing the systematic and scientific approach of scoping review. On the other hand, the weakness of this paper would be the lack of critical thinking around the meaning of this finding and how this can change in the future.\n\nThe rationale, and objectives of, the Systematic Review were clearly stated. The detailed process of the scoping review was clearly described, and the conclusion was adequately supported by the results presented in the review.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? 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": "295677", "date": "08 Jul 2024", "name": "Luca Giuseppe Re", "expertise": [ "Reviewer Expertise Clinical Nursing Pediatric Nursing Systematic Review Meta-analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI thank the Editor for the opportunity to review this very interesting study, which concerns a topic very dear to me that has returned to the fore during the period of the COVID-19 pandemic. In my opinion, however, for indexing of the manuscript to be possible, it is necessary to overcome some methodological limitations that emerged after careful reading. I've included my notes below.\nAbstract Methods Section I would suggest the authors move the part starting with “After Removal 63 Duplicates (…)” and ending with “so 17 Studies (…)” to the beginning of the Results section\nMain text Methods Section Subsection Research Methods I would kindly ask the authors to report the day, month and year in which (or starting from which) they carried out the query of the biomedical databases Subsection Study selection Also in this case, I would suggest the authors to move the part that begins with “The initial search produced a total of 1,005 articles (…) and ends with “(…) therefore 17 studies were finally analyzed (see Figure 1)” to beginning of the Results section, as well as Figure 1. I would suggest the authors move Table 2 to the Results section to the subsection “General characteristics of the included studies”\nDiscussion section Subsection Personal characteristics The authors illustrate the contents that emerged from the documents they found, but they should also express their own considerations, a typical characteristic of the Discussion section Subsection Limitations I would suggest the authors highlight two other important limitations: a) they only included documents in English (this limitation is important, in reference to the fact that, for example, the COVID-19 pandemic has had a great impact on moral distress of intensive care nurses around the world, hence the flourishing of many studies in languages other than English); b) they did not query a biomedical database relevant to the topic, such as PsycINFO, and did not consult gray literature resources\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [ { "c_id": "11994", "date": "10 Jul 2024", "name": "Amina Mussa Ahmad", "role": "Author Response", "response": "Dear Dr. Luca, Thank you for the opportunity to review our study and for your valuable comments. Your feedback has significantly enhanced the readability and quality of our article. We deeply appreciate your thorough review and insightful suggestions. We are pleased to inform you that almost all points raised in your review have been addressed and modifications have been made accordingly in the revised version of the study. Below is a summary of the changes made: Note 1: Abstract-Methods Section The suggested part has been moved to the Results section. Note 2: Methods Section-Subsection Research Methods The month and year from which the search was initiated have been added to the Methods section, specifically highlighting that the search started in July 2022. Note 3: Subsection Study Selection The specified part has been moved to the beginning of the Results section as suggested. As for Table 2, we have chosen to keep it in the Data Analysis section as it is intended to be the data extraction table that summarizes the findings of the results, which are illustrated in Table 3. Note 4: Discussion Section-Subsection Personal Characteristics We have added a discussion of the findings along with the authors' assumptions and considerations, as you recommended. Thank you for this valuable note. Note 5: Subsection Limitations We have considered your valid points and added the mentioned limitations to the Limitation section, acknowledging the exclusion of non-English documents and the lack of querying certain relevant biomedical databases and gray literature resources. Once again, thank you for your constructive feedback and for helping us improve our study." } ] }, { "id": "295684", "date": "15 Jul 2024", "name": "Ratchaneekorn Upasen", "expertise": [ "Reviewer Expertise My area is mental health and psychiatric 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\nThank you very much for giving me to comments and suggests this manuscript. There are some comments, please see as below: 1. In the abstract mentions relevant studies published between 2011 to 2022, but the text states that ..articles published in the period between January 2010 to July 2022... please check again 2. Study selection part, please provide key words to search each data based 3. Data analysis section, please explain more how to analyze the qualitative data? which was method used in this study? 4. The qualitative studies just present theme or categories about related factors to moral distress in ICU nurses, so the data was not tested that associated or related to the moral distress. How to make sure that they are related factors or associated the moral distress. 5. Sub theme \"Nurse autonomy\" How to related to the major theme \"powerlessness\" Please explain in discussion 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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-1574
https://f1000research.com/articles/13-219/v1	25 Mar 24	{ "type": "Study Protocol", "title": "A study on health care utilization and out of pocket expenditure in rural central India: A cross-sectional study", "authors": [ "Ankita Hepat", "Dr. Swarupa Chakole", "Dr. Swarupa Chakole" ], "abstract": "Background Out-of-pocket health expenditures have significant adverse effects, potentially resulting in impoverishment and impacting the quality of life. Awareness of patterns of healthcare utilization and out-of-pocket expenses is essential for informing healthcare scheme decisions. According to estimates from the World Health Organization in 2005, illness and the financial strain of healthcare costs force 25 million households into poverty each year. The goal of the study is to assess the proportion of households incurring out-of-pocket expenditure (OOPE) and The mean quality expended by households on healthcare.\n\nMethods In Wardha district, a cross-sectional study will be conducted. The pre-tested semi-structured survey will be administered to examine a sample of adult age groups, to identify sociodemographic data, utilization of healthcare services, and OOPE. A sample of 246 participants was selected by random sample method.\n\nConclusion This study will help to improve and assess healthcare utilization and reduce out-of-pocket expenditures that lead to catastrophe and impoverishment. Awareness of the participants about health insurance. And they were known about the healthcare services.", "keywords": [ "Health care utilization", "Out-of-pocket expenditure", "Health care coverage", "Health insurance", "Co-morbidities." ], "content": "Introduction\n\nUniversal access to high-quality healthcare services is available to everyone in the community, including promotive, preventive, curative, rehabilitative, palliative services, and mental impairment in people without any financial difficulties.1,2 The High-Level Expert Group (HLEG) on Universal Health Coverage proposes the performance of a “National Health Package,” encompassing comprehensive health services that will be provided to every individual free of cost.3 Appropriate planning and implementation of healthcare services requires reliable data on trends in morbidity and mortality, also trends in the utilization of healthcare facilities are considered. The socioeconomic status of the population depends on the healthcare utilization services, the disease severity, and the availability, accessibility, and affordability of healthcare services.4 The National Health Accounts (2016) in India households that contribute 64.2% of the Total Health Expenditure (THE) through out-of-pocket payments. Although the burden of out-of-pocket expenditure (OOPE) decreased from the 2004 to 2005 estimate (comprising 71% of the total health expenditure), it remains unsatisfactorily high.5\n\nGlobally, the primary objective of healthcare systems is to establish suitable funding mechanisms, ensuring individuals can access preventive and curative health services without falling into poverty.6\n\nThe reason for the health system is to enhance access to healthcare and utilization of resources by making them more affordable and to distribute the cost of healthcare services among every individual in a risk pool to decrease the economic burden of illness. however, the insurance penetration rate remains low in India, attributed to either a lack of knowledge or the functional status of the existing health insurance schemes.7\n\nThe Sustainable Development Goals (SDGs) were completed in September 2015, expanded, and build upon the Millennium Development Goals (MDGs) by incorporating updated targets. Derived from the principle of “leaving no one behind,” the Sustainable Development Goals (SDGs) consist of 17 goals and 169 targets. There are 17 goals in total, with Goal No. 3 specifically dedicated to health. SDG 3 comprises 13 targets, three initiatives are dedicated to improving maternal and child health, while another three focus on addressing both communicable and non-communicable diseases as well as addiction. Additionally, two initiatives target environmental health, and one aims to achieve Universal Health Coverage (UHC). The remaining four objectives encompass the implementation of tobacco control policies, vaccination and medication strategies, health financing, workforce development, and preparedness for global health risks.8\n\nIn India, health-related public expenditure is managed by three tiers of the government: the Central Government, the State Government, and the local bodies.9 Although government businesses and insurance providers are major healthcare funding agencies, each family and individual covers each of the healthcare expenses that can be incurred through OOP payments, insurance premiums, or government taxes.4 In India, Gross Domestic Product (GDP) spends only 5% on health and 80% of this takes the form of OOPE in rural areas. Also, rural households designated 3.31% (ranging from 0.4% to 10.96%) of their household budget for out-of-pocket expenditure, which is high in rural areas.3,5 In a prior investigation examining estimates on worldwide catastrophic spending and out-of-pocket (OOP) payments, it was found that around 150 million individuals experience a financial burden annually due to healthcare expenses. Additionally, approximately 100 million individuals fall into poverty as an outcome of OOP payments.6\n\nChronic health conditions have increased globally as a result of an unequaled increase in risk factors for non-communicable diseases (NCDs). The interaction and tendency to cluster among these NCDs result in the occurrence of comorbidity (the simultaneous presence of two or more NCDs), without a distinct primary index disease. The circumstances in middle-class and low-income nations are comparable. A systematic review revealed that the incidence of comorbidity in South Asia ranges between 4.5% and 83%. The analysis also indicated that the most frequently reported outcomes were decreased physical functioning and increased healthcare. Moreover, in developing Nations, healthcare is predominantly funded through OOP payments, and households facing a significant limitation on their scheme.10\n\n\nRationale\n\nThe previous study indicated that the median total OOPE for incurable diseases was determined to amount to 5000 Indian Rupees. Additionally, it was noted that 41.6% of the participants’ overall earnings were allocated to healthcare expenses related to chronic diseases. This percentage suggests a situation of catastrophic health expenditure. Notably, health insurance coverage remains at a low level, and the utilization of social assistance for treatment is minimal.9\n\nThis study will be conducted because in-patient and out-patient care are associated with the highest out-of-pocket expenditure and less information about healthcare utilization patterns. Only 20 percent of households in Maharashtra have any kind of health insurance that covers at least one member of the household. This study will also fill the knowledge gap in Maharashtra people who are unaware of healthcare utilization patterns and OOPE in rural areas.\n\nTo assess healthcare utilization and out-of-pocket expenses among the rural population.\n\n\n\n1. Association between socio-demographic variables and health care utilization services among adults in rural Wardha.\n\n2. To identify the utilization pattern of healthcare services of the people living in rural areas.\n\n3. To identify the health insurance coverage.\n\n4. To assess the proportion of households experiencing out-of-pocket expenses and the average expenditure incurred by each household for healthcare.\n\n\nMethod\n\nThe current study will be a cross-sectional study.\n\nThe study will be conducted within the Wardha District of Maharashtra, specifically in the rural field practice area, under the Datta Meghe Institute of Higher Education and Research (DMIHER) Wardha.\n\nThe study will be conducted from January 2024 to May 2024.\n\nThe present study will include the adults age (25 to 59 years), both males and females in rural areas. In Wardha district.\n\nInclusion criteria:\n\n1. Adults age group (25 to 59 years) people who suffered from any health problem since 1 year and above\n\n2. Individuals who are willing to participate.\n\nExclusion criteria:\n\n1. Those who are mentally unstable.\n\n2. Serious ill participants are excluded.\n\n3. Those adults who are healthy and do not have any illness.\n\nThe prior research study, which was utilized as a mother research study, found that the higher prevalence of out-of-pocket expenditure was 80% in rural areas. The following articles were used to estimate the number of participants for this investigation.\n\nWith the help of the following formula, the sample size was calculated:\n\nEstimated proportion (p): 0.8\n\nEstimated error (d): 0.05\n\nAlpha (α): 0.05\n\nSample size: 246\n\nTherefore, a total of 246 adults will be included in the sample size to collect data and assess healthcare utilization and OOPE in rural areas.\n\nThe study will be conducted in the Wardha district, and only 1 village selected for data collection by using a simple random sampling method. After the selection of the village, 246 households will be selected and will be interviewed for data collection. Data will be collected using pre-tested semi-structured questionnaires. The Kobo toolbox will be used.\n\n\n\n1. Socio-demographic variables\n\nIt includes gender, religion, economic status, family types, and type of house.\n\n2. Healthcare utilization for diseases\n\nIt includes the system of medicine, type of service, type of hospital, type of government hospital, and type of insurance availed by the participant.\n\n3. Out-of-pocket expenditure\n\n• The data collected the OOPE of the study participant due to chronic morbidity. Variables include Monthly hospitals expenses, charge a pay in diagnostic/investigation.\n\nData will be collected using pre-tested semi-structured questionnaires. The Kobo Toolbox will be used for the data collection procedure. The questionnaires will be created using the Kobo toolbox. This questionnaire will have three sections, first is sociodemographic data (e.g. gender, religion, economic status, family type, and type of house), second is health care utilization in rural areas and third is out-of-pocket expenditure of adults in rural areas. The tool’s objective is to assess the healthcare utilization and OOPE of adults in rural areas. A written consent form will be taken participants before participate in the study. Variables, data sources and method of data collection shown in Table 1.\n\n\n\n• Gender\n\n• Religion\n\n• Economic status\n\n• Family type\n\n• Type of house\n\n\n\n• Types of diseases\n\n• Types of hospitals\n\n• System of medicine\n\n• Type of service\n\n• Type of government hospital\n\n• Type medicine insurance availed by the participant\n\n• Awareness of health insurance\n\n\n\n• Monthly hospitals expenses\n\n• Charge a pay in diagnostic/investigation\n\n• Monthly medical expenses\n\nData will be collected in pretested semi-structured questionnaires. The purpose of the interview will be to explain to the participants in their local language so they can understand the objective of the study properly and be willing to participate in the study. Information will be gathered through interviews during house-to-house visits. To identify the healthcare utilization patterns and OOPE in rural areas. The demographic questionnaires, healthcare utilization patterns, and OOPE questions set in the Kobo Toolbox software (https://www.kobotoolbox.org/) it will be used for data collection during the interview.\n\nRecall bias may occur as participants may not accurately remember or report their healthcare utilization or out-of-pocket expenditure. This can affect the reliability of the data.\n\nSelection bias can occur when the sample of individuals included in a study is not representative of the larger population. Random sampling techniques aim to eliminate bias by ensuring that every member of the population has an equal opportunity to be included in the study.\n\nThe Datta Meghe Institute of Higher Education and Research (Deemed to be University). Institutional Ethics Committee approval for this study protocol Ref. No. DMIHER (DU)/IEC/2023/37. Date: 20/12/2023. Additionally, written informed consent will be obtained from all the participants before participating in the study. Privacy and confidentially will be maintained throughout the study. Measures will be taken to minimize any potential harm a risks to the participants.\n\nThe data will be entered in Microsoft Excel. This data will be encoded and entered, and the data will be analyzed using R Statistical software 4.3.2 version (https://www.r-project.org/). The data will be tabulated and visualized through graphs and tables. Inferential statistics like T-tests and chi-square tests will be used.\n\nThe research is not yet started. The proposal of this study was submitted for ethical approval to the IEC department of DMIHER (DU) and approved by the ethical committee.\n\nThis study will improve and assess the healthcare utilization and OOPE in a rural area and who will get the proportion of utilization services according to the co-morbidity, and health insurance card. We will get some significant factors regarding health care utilization and services.\n\n\nDiscussion\n\nHarish et al. (2020) have done a cross-sectional in Kerala. The goal of the study is to find the health insurance coverage and its impact on out-of-pocket (OOP) expenditure in rural areas. Health insurance coverage was determined to be 74%. A quarter of patients lack insurance coverage. While all patients face out-of-pocket (OOP) expenses, insured individuals notably incur lower OOP costs.7\n\nDalal K et al. (2017) This study was conducted a household survey in Cambodia. This analysis explores the household impacts of out-of-pocket (OOP) healthcare payments, focusing on their catastrophic, economic, and fairness aspects. The finding indicated inequality and unfairness in healthcare payments, with a higher prevalence of catastrophic spending among the impoverished.6\n\nVasudevan et al. (2019): A cross-sectional study was conducted in Puducherry. To assess the percentage of households incurring out-of-pocket expenses and the mean expenditure per household on healthcare. The study included 240 households, evenly divided between 120 rural and 120 urban. According to this study, the prevalence of households incurring out-of-pocket healthcare expenses was 68.3% in rural areas, while in urban areas, it was 65.8% (ranging from 57% to 73.7%).5\n\nMathumkunnath Vijayan et al. (2020) performed a cross-sectional study in Thrissur district of Kerala. The goal of the study is to understand the patterns of healthcare utilization patterns and out-of-pocket expenditures that are crucial for healthcare policy-making. 26.6% of the study participants had obtained health insurance. Health insurance coverage is minimal, and the utilization of community assistance for treatment is limited. These factors result in high OOP expenses, reaching the level of catastrophic health expenditures.9\n\nThuong et al. (2020) Household Living Standard Surveys in Vietnam is a Southeast Asian. To investigate the effect of these health insurance initiatives on the utilization of healthcare facilities and out-of-pocket health expenditures (OOP). We establish that Health insurance has raised the expectation of seeking outpatient care, the overall total outpatient visits, total visit count, and the mean number of visits at the district level of healthcare providers. The health insurance scheme increased the healthcare utilization services and concurrently reduced out-of-pocket expenses for the participants.2\n\nIn 2022 Karan et al., the National Sample Survey Organization (NSSO) 75th round was organized. This study aims to produce current evidence regarding the economic implications of comorbidity on households, particularly regarding OOPE, and their consequences on experiencing catastrophic out-of-pocket expenditure. The findings of this study indicated that the outpatient OOPE is consistently lower when comorbidity is present, in comparison to situations involving single conditions. This study concludes that, the multimorbidity results in elevated and catastrophic out-of-pocket payments by households.10\n\nFelix Masiye (2016) a cross-sectional dataset from the Zambian Household Health Expenditure and Utilisation Survey (ZHHEUS). To investigate the factors associated with healthcare choices among individuals who are ill. The conclusion drawn from this study the population’s access to healthcare is strongly influenced by their socio-economic status, type of illness, and region of residence.11\n\nMost of the individuals are unable to afford the treatment for chronic diseases on their own and fall into debt. Identifying patterns of healthcare utilization can help in understanding the existing gaps in healthcare access. The results of the study can be used to advocate for the improvement of healthcare infrastructure in rural areas and ensure the population has adequate and accessible healthcare facilities. In rural areas of India, there is a need to enhance awareness among adult individuals regarding the free healthcare facilities provided by the government.\n\n\n\n• Data will be collected by face-to-face interviews, which may affect the accuracy of the result. The study may be affected by recall bias.\n\n• This data lacks information regarding the sums acquired through numerous financing sources, therefore the amount of hardship financing could not measured.\n\nThe study protocol will be published in an indexed journal and presented at seminars and conferences.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nRepository Name: Figshare\n\nFile Name: STROBE Checklist for “A study on health care utilization and out of pocket expenditure in rural central India: A cross-sectional study”, DOI: 10.6084/m9.figshare.25342510.v2. 12\n\nLicence: CC BY 4.0\n\n\nReferences\n\nHealth care – Wikipedia: [cited 2023 Aug 21]. Reference Source\n\nThuong NTT, Huy TQ, Tai DA, et al.: Impact of health insurance on health care utilisation and out-of-pocket health expenditure in Vietnam. Biomed Res Int. 2020 Aug 26; 2020: 1–16. Publisher Full Text\n\nArchana R, Kar SS, Premarajan K, et al.: Out-of-pocket expenditure among the households of a rural area in Puducherry, South India. J Nat Sci Biol Med. 2014; 5(1): 135–138. PubMed Abstract \| Publisher Full Text\n\nRay TK, Pandav CS, Anand K, et al.: Out-of-pocket expenditure on healthcare in a north Indian village.\n\nVasudevan U, Akkilagunta S, Kar SS: Household out-of-pocket expenditure on health care - A cross-sectional study among urban and rural households, Puducherry. J Fam Med Prim Care. 2019 Jul; 8(7): 2278–2282. Publisher Full Text\n\nDalal K, Aremu O, Ussatayeva G, et al.: Out-of-pocket health expenditure and fairness in utilization of health care facilities in Cambodia in 2005 and 2010. F1000Res. 2017 [cited 2023 Aug 11]. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nHarish R, Suresh RS, Rameesa S, et al.: Health insurance coverage and its impact on out-of-pocket expenditures at a public sector hospital in Kerala, India. J Fam Med Prim Care. 2020 Sep; 9(9): 4956–4961. Publisher Full Text\n\nAskari MH, Gupta K: Understanding the health care utilization behavior to achieve the sustainable development goals—a comparative study of Malda District, India. SN Soc Sci. 2022 Sep 1; 2(9): 1–22. Publisher Full Text\n\nMathumkunnath Vijayan S, Puliyakkadi S, Chalil S: Health care utilization and out of pocket expenditure in a rural area of Kerala: a cross sectional study. Int J Community Med Public Health. 2020 Jul 24; 7: 3081. Publisher Full Text\n\nKaran A, Farooqui HH, Hussain S, et al.: Multimorbidity, healthcare use and catastrophic health expenditure by households in India: a cross-section analysis of self-reported morbidity from national sample survey data 2017–18. BMC Health Serv Res. 2022 Sep 12; 22(1): 1151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasiye F, Kaonga O: Determinants of healthcare utilisation and out-of-pocket payments in the context of free public primary healthcare in Zambia. Int J Health Policy Manag. 2016 Jun 1; 5(12): 693–703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHepat A: STROBE Checklist for “A study on health care utilization and out of pocket expenditure in rural central India: A cross-sectional study”. figshare. Online resource. 2024. Publisher Full Text" }	[ { "id": "282567", "date": "24 Jun 2024", "name": "Sinan Erdoğan", "expertise": [ "Reviewer Expertise Health economics", "Sustainable development goals" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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) There are some critical mistakes in the study. For instance, authors state that the Sustainable Development Goals (SDGs) were completed in September 2015. However, SDGs have not completed yet, instead, they are in effect. 2) Theoretical discussions and study rationale ignore recent papers in the field. Therefore, there is a need for revision, and the following study may give ideas on expanding the theoretical discussions: Analyzing the asymmetric effect of disaggregated health expenditures on economic growth 3) There are some minor grammatical issues in the study. 4) Abbreviations should not be used in the abstract section. 5) I would like to see research results and empirical outcomes.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "11998", "date": "15 Jul 2024", "name": "Ankita Hepat", "role": "Author Response", "response": "1) There are some critical mistakes in the study. For instance, authors state that the Sustainable Development Goals (SDGs) were completed in September 2015. However, SDGs have not completed yet, instead, they are in effect. Response: Added the recent SDG goal, correction done 2) Theoretical discussions and study rationale ignore recent papers in the field. Therefore, there is a need for revision, and the following study may give ideas on expanding the theoretical discussions: Analyzing the asymmetric effect of disaggregated health expenditures on economic growth Response: The rationale and discussion are expanded according to the question. 3) There are some minor grammatical issues in the study. Response: All Grammarly is corrected 4) Abbreviations should not be used in the abstract section. Response: Correction is done. 5) I would like to see research results and empirical outcomes. Response: Yes mam/ sir" } ] } ]	1	https://f1000research.com/articles/13-219
https://f1000research.com/articles/12-802/v1	10 Jul 23	{ "type": "Research Article", "title": "Weekly paclitaxel, carboplatin and cetuximab (PCC) combination followed by nivolumab in platinum-sensitive recurrent and /or metastatic squamous cell carcinoma of head and neck: a retrospective analysis from two institutions in India", "authors": [ "M V Chandrakanth", "Vivek Agarwala", "Pradip Mondal", "Raajit Chanana", "K M Parthasarathy", "Sourav Dutta", "Harsh Dhar", "Suman Mallick", "Sayan Das", "Devmalya Banerjee", "Md Arif Faizan", "Moinak Basu", "Subhabrata Kumar", "M V Chandrakanth", "Pradip Mondal", "Raajit Chanana", "K M Parthasarathy", "Sourav Dutta", "Harsh Dhar", "Suman Mallick", "Sayan Das", "Devmalya Banerjee", "Md Arif Faizan", "Moinak Basu", "Subhabrata Kumar" ], "abstract": "Background: First line (1L) TP-Ex-like regimen followed by 2nd-line (2L) immunotherapy represents one of the standards of care in platinum-sensitive recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M SCCHN). We report our experience from 2 tertiary care institutions of India. Methods: This is a retrospective analysis of consecutive patients of platinum-sensitive R/M SCCHN treated with 1L weekly paclitaxel, carboplatin, and cetuximab (PCC) regimen followed by cetuximab maintenance (if non-progressive) or 2L nivolumab or oral metronomic chemotherapy (OMCT) on progression. Overall response rates (ORR), progression-free survival after 1L and 2L (PFS-1 & PFS-2), overall survival (OS), and safety were evaluated. Results: The study included 54 patients; median age 56.5 years; 89% men; 11% had cardiac dysfunction; 13% had renal dysfunction. After 1L PCC, ORR was 59.3%; median PFS-1 was 7.031 months; 61% had progression; 35% were treated with nivolumab and 18% with OMCT. The ORR was 26.3% (nivolumab) and 10% (OMCT). Median PFS-2 was 6.5 months (nivolumab) and 2 months (OMCT). The median OS was 15.01 months (entire cohort), 20.6 months (nivolumab), and 7 months (OMCT). Grade III/IV adverse events on PCC included neutropenia (31.4%), anaemia (35.1%), thrombocytopenia (7.4%), febrile neutropenia (11.1%), and skin reaction (16.6%); no Grade-III/IV treatment-related toxicities on 2L. Conclusions: 1L weekly PCC is an effective regimen for palliative therapy of platinum- sensitive R/MSCCHN with an acceptable toxicity profile. The addition of 2L nivolumab on progression further improves the outcomes.", "keywords": [ "Palliative therapy", "Cetuximab", "Nivolumab", "Immunotherapy", "Paclitaxel", "Carboplatin", "Chemotherapy." ], "content": "Introduction\n\nSquamous cell carcinomas of head and neck (SCCHN) accounts for 90% of all head and neck cancers (HNCs)1,2 The cumulative risk of developing HNC (oral and pharynx) in India is 1 in 60, with males at higher risk than females (cumulative risk 1 in 41: 1 in 112).3 Approximately 66% of HNCs in India are diagnosed at a locally advanced (LA) stage.3 Though, a disease of sixth or seventh decade of life, HNC is increasingly being seen in younger population.1\n\nSCCHN is a difficult to treat cancer, especially in India, due to difference in genetic and etiological factors, in additional to logistic issues, compared to western world.2 The etiology of HNC in India is primarily driven by smokeless tobacco chewing versus human papilloma virus (HPV) infection seen in western world.2 In recurrent and/or metastatic (R/M) setting, treatment of SCCHN is even more difficult with poor outcomes.\n\nHistorically, in R/M SCCHN, combination chemotherapy regimens such as platinum plus 5-fluorouracil (5-FU)4–7 and platinum plus taxane8–14 significantly improved response rates compared with single agent chemotherapy, but they were not successful in demonstrating an improved OS and showed higher toxicity.15 Cetuximab based combinations with platinum-based doublet chemotherapy such as the EXTREME regimen (cetuximab-cisplatin/carboplatin-5-fluorouracil [5-FU])16,17 or the TPExtreme regimen (cetuximab-docetaxel-cisplatin),18 followed by maintenance cetuximab until disease progression, showed increased OS compared with doublet chemotherapy and became the best 1L approach in R/M SCCHN.19,20 Also the EXTREME regimen is associated with hematological toxicities that raised the need for safer regimens.21 The CEMET trial showed that paclitaxel, carboplatin and cetuximab (PCC) regimen is a feasible 1L treatment option and is associated with significantly lesser toxicities than the EXTREME regimen (40% vs. 60%; p = 0.034).22\n\nMany patients with R/M HNSCC show disease progression after 1st line platinum-based therapies.23 In these patients, the U.S. Food and Drug Administration (FDA) has approved PD-1 inhibitors like nivolumab and pembrolizumab for 2nd line treatment.24,25\n\nAfter the success of the Keynote-048 study,26 1st-line immuno-chemotherapy (IC) with pembrolizumab-platinum-5FU combination (irrespective of programmed death-ligand 1 [PD-L1] status) or immunotherapy alone with pembrolizumab (for PD-L1 ≥ 1%) became the preferred option of systemic therapy for platinum sensitive R/M SCCHN.15 However, the EXTEME and TP-Extreme regimens, still remain one of the standard first-line options in R/M SCCHN, especially if immunotherapy is not feasible, in PD-L1 negative patient population, in oral primary, and in rapidly progressive bulky disease which requires faster responses.18,23,25,27,28\n\nWith increasing treatment options in R/M SCCHN, sequencing becomes important. However, sequencing has not been addressed well in the clinical trials. In KN -048 study, only 16.7% patients in cetuximab arm received subsequent immunotherapy, while in IC or immunotherapy arm only 16.4% patients received subsequent EGFR inhibitor.29 So, the right sequencing of therapies in RM SCCHN is still elusive.\n\nHere we report our experience with 1st-line weekly PCC combination followed by 2nd-line immunotherapy (nivolumab) in platinum-sensitive R/M SCCHN from two tertiary care centers of India.\n\n\nMethods\n\nThis study involved a retrospective analysis of consecutive patients with platinum-sensitive R/M SCCHN treated between August 2017 and May 2020 at the two tertiary care centres in India with 1st-line weekly combination paclitaxel 80 mg/m2, carboplatin AUC2 and cetuximab 400 mg/m2 loading followed by 250 mg/m2 (PCC) for a maximum duration of 12 weeks. Non-progressive patients were then started on cetuximab maintenance. Patients were further treated with 2nd-line nivolumab or oral metronomic chemotherapy (OMCT) on progression as per feasibility. Eligible patients 18–70 years old with ECOG-PS 0-2 and had at least one measurable lesion as per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) were included. Exclusion criteria included any previous systemic chemotherapy for HNSCC surgery or radiotherapy within the previous 6 weeks; a previous dose of cisplatin more than 300 mg/m2; treatment with EGFR-targeting therapy within the previous 12 months; clinically significant cardiovascular disease; other malignancies within 5 years before randomisation, Written consent was obtained from the patients.\n\nDemographic, clinical and treatment characteristics data were collected from patients’ charts until July 7, 2020. Response evaluation was performed as per RECIST 1.1 (for solid tumors) after every 8–12 weeks.30 Toxicities during treatment were documented in accordance with CTCAE version 4.03.31 The institutional ethics committees of the Narayana Superspecialist Hospital, Howrah and Dharamshala Narayana Superspecialist Hospital, New Delhi approved the study. Written informed consent was obtained from each patient.\n\nTo evaluate the overall response rates (ORR), progression-free survival, and overall survival (OS) after 1L PCC and 2nd line therapies. The study also evaluated the safety of 1L PCC and second-line therapies.\n\nStatistical measures were calculated using software SPSS Version 20. PFS and OS were estimated by Kaplan-Meier method and compared by log rank test. Median follow-up was calculated using the reverse Kaplan Meier method. Mean ± S.D. and Median were used to summarize the quantitative variables.\n\n\nResults\n\nFifty-four patients with R/M SCCHN were treated at the two centers between August 2017 and May 2020. Median age of the study population was 56.5 years; 89% were males. Oral cancer was the primary cancer site in 30 (55%) patients and 24 (45%) had non-oral primary cancer sites. Of the 54 patients, 16 (30%) were treatment naïve; 38 (70%) had received prior treatments (chemotherapy or surgery or radiation or a combination of these) and had a median treatment free interval (TFI) of 10 months; 28 (51%) had not received any previous systemic therapy. A history of tobacco abuse was present in 41 (75%) of patients. All patients had Eastern Cooperative Oncology Group performance status (ECOG PS) of 1 or 2 at the start of PCC; 6 (11%) had cardiac dysfunction and 7 (13%) had renal dysfunction. Demographic details of the study population are provided in Table 1.\n\nThe study schema is outlined in Figure 1. All patients received 1L weekly combination of paclitaxel 80 mg/m2, carboplatin AUC2 and cetuximab 400 mg/m2 loading followed by 250 mg/m2 (PCC) for up to maximum 12 weeks, followed by weekly cetuximab maintenance 250 mg/m2until disease progression or intolerable adverse effects. Thirty-three (61%) patients progressed on 1L PCC and 21 (39%) did not. The patients who progressed on 1L PCC, were further treated with second line (2L) nivolumab 3 mg/kg two weekly (n = 19) or oral metronomic chemotherapy (n = 10) (OMCT – celecoxib 200 mg twice daily + erlotinib 100 mg once daily + methotrexate 12 mg/m2 once weekly) as per feasibility. Four patients who progressed on 1L PCC were managed on best supportive care (BSC) alone. Non-progressive patients were given single-agent cetuximab in maintenance dose.\n\nThe median number of Cetuximab cycles used as 23. Outcomes of the study population in 1L PCC is given in Table 2\n\nThe best response after second line therapies is summarized in Table 3. The ORR was 26.3% with nivolumab and 10% with OMCT. Median PFS and OS after second line therapy were 6.5 months and 20.6 months with nivolumab, whereas 2 months and 7 months with OMCT, respectively (Figure 2).\n\nAfter a median follow-up of 21 months, 30 patients were alive; 21 on 1L PCC who were on cetuximab maintenance and nine on 2L nivolumab; 24 succumbed to the disease.\n\nFirst line PCC was generally well tolerated. Grade III/IV adverse events on 1st-line PCC included neutropenia (31.4%), anemia (35.1%), thrombocytopenia (7.4%), febrile neutropenia (11.1%) and skin reaction (16.6%). There were no grade III/IV treatment related toxicities in second line therapies.\n\n\nDiscussion\n\nCurrent study shows that weekly paclitaxel-carboplatin appears as an equally effective backbone for cetuximab with acceptable safety profile as compared to EXTREME regimen (cisplatin-5FU)16,17 or TPExtreme regimen (docetaxel-platinum)18 in 1st-line platinum sensitive R/M HNSCC (Table 3). Further, 2nd line immunotherapy after 1st line PCC is shown to significantly improve outcomes in comparison with OMCT.\n\nIn our study, 1st-line PCC had a good efficacy profile in R/M SCCHN with ORR of 59.3%, and PFS and OS of 7.03 months and 15.01 months. In the EXTREME regimen 1st-line cetuximab plus chemotherapy significantly prolonged median OS compared to chemotherapy alone (10.1 versus 7.4 months; hazard ratio [HR] for death 0.80, 95% CI 0.64-0.99). Median PFS (5.6 months versus 3.3 months) and ORR also improved significantly (36% versus 20%).17 A phase III trial compared EXTREME regimen with cetuximab plus cisplatin and docetaxel (TPExtreme regimen).18 After a median follow-up of 30 months, the median OS of the EXTREME and docetaxel based TPExtreme regimen was comparable (14.5 months versus 13.4 months). The TPExtreme regimen did not look at PFS and ORR. However, in the phase II GORTEC study, cetuximab plus cisplatin and docetaxel resulted in an ORR of 51.9% and PFS and OS of 6.2 and 14 months, respectively (Table 3).20 The TPExtreme regimen was more tolerable than EXTREME with lower grade ≥3 adverse events (34% versus 50%) and had more patients initiating maintenance cetuximab than EXTREME regimen (73% versus 53%). In our study also, the PCC regimen had an acceptable safety profile that was safer than EXTREME and comparable to TPExtreme.\n\nOur results of efficacy and safety of 1st line PCC are in tandem with other data on 1st-line PCC like CSPOR-HN0232 and CACTUX33 study (Table 4). The CSPOR-HN02 study which used split dose paclitaxel (100 mg/m2 on days 1 and 8 every three weeks) showed that the PCC regimen can be given on an outpatient basis, thereby reducing cost of treatment.21 In our study also, PCC was given on outpatient basis. This is an important aspect to consider in a country like India as it significantly reduces cost of treatment and need for admission beds. However, the CSPOR-HN02study did not look at treatment after progression on 1st line PCC. On the other hand, only 22% of patients in the CACTUX trial received PD-1 inhibitor immunotherapy after progression and demonstrated a: median PFS of 2.2 months.33 Comparatively, in our study, 19 of 33 patients who progressed (57.5%) received immunotherapy (nivolumab) and showed good response with an ORR of 26.3% and median PFS and OS of 6.5 months and 20.6 months, respectively.\n\n* Full analysis of adverse events were not available at the time of this publication.\n\nThough EXTREME regimen continues to be the 1st-line standard of care in R/M SCCHN, many patients are ineligible for cisplatin-based regimen primarily due to renal and cardiac dysfunction.34,35 We found that PCC was safe and well tolerated even in patients with cardiac and renal dysfunction. Patients in our study could receive median of 23 cycles of Cetuximab based therapy.\n\nWe do note that early immunotherapy has shown OS advantage in KN48 study26 and that the PFS-2 of 11months reported is superior to that in our study. However, KN-48 data may not be applicable in patients of Indian subcontinent. Unlike Indian patients, who predominantly have oral cavity primaries and mainly tobacco related etiology, KN-48 patients had mainly non-oral primaries and HPV related etiology. Other barriers to the ease of using immunotherapy front line are the accessibility to PDL-1 combined positive score testing and its relatively higher cost. Also, the OS results in the Keynote -048 study are mainly driven by the PD-L1 combined positive score>20 subgroup.\n\nAnother matter of concern is that, more than 1/3 patients in the pembrolizumab monotherapy group may have frank disease progression in the initial three months of therapy. This is more worrisome in Indian patients because they have a far higher disease burden than that of the western population. Hence, clinicians may lose this narrow window of opportunity and the patients may not remain eligible for subsequent anticancer therapy in view of rapid disease progression and deteriorating performance status. Hence response rate become a more important endpoint in Indian patients and may act as a surrogate to better quality of life and survival. However, a randomized prospective trial comparing the alternate sequencing of immunotherapy-based and cetuximab-based therapy in R/M HNSCC will be able to better guide the efficacy, safety and sequencing of these drugs.\n\nThe study is limited by its retrospective design and small patient population. Additionally, PD-L1 expression was not performed, so we cannot access which patient population actually benefited from immunotherapy. Despite this our study has contributed immensely to the growing body of knowledge that cetuximab based PCC regimens can be used effectively and safely in 1st-line palliative care of R/M SCCHN. To the best of our knowledge, this is the first real-world data of 1st-line PCC followed by immunotherapy from Asia.\n\n\nConclusions\n\nFirst-line weekly PCC is an effective regimen for palliative therapy of platinum-sensitive R/M SCCHN with acceptable toxicity profile. Addition of 2nd-line nivolumab on progression, further improves the outcomes.", "appendix": "Data availability\n\nFigshare: TP Ex for analysis- Weekly paclitaxel baseline data- Revised.xlsx, https://doi.org/10.6084/m9.figshare.22657699.v1. 36\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe writing of this article was supported by a medical writer at Mediception Science Pvt Ltd.\n\n\nReferences\n\nSharma JD, Baishya N, Kataki AC, et al.: Head and neck squamous cell carcinoma in young adults: A hospital-based study. Indian J. Med. Paediatr. Oncol. 2019 Jul 1; 40(5): S18–S22. Publisher Full Text\n\nPrabhash K, Babu G, Chaturvedi P, et al.: Indian clinical practice consensus guidelines for the management of squamous cell carcinoma of head and neck. Indian J. Cancer. 2020 Feb 1; 57(5): 1–S5. PubMed Abstract \| Publisher Full Text\n\nMathur P, Sathishkumar K, Chaturvedi M, et al.: Cancer Statistics, 2020: Report From National Cancer Registry Programme, India. JCO Glob. Oncol. 2020 Jul 16; 6: 1063–1075. PubMed Abstract \| Publisher Full Text\n\nForastiere AA, Metch B, Schuller DE, et al.: Randomized comparison of cisplatin plus fluorouracil and carboplatin plus fluorouracil versus methotrexate in advanced squamous-cell carcinoma of the head and neck: a Southwest Oncology Group study. J. Clin. Oncol. 1992 Aug; 10(8): 1245–1251. PubMed Abstract \| Publisher Full Text\n\nJacobs C, Lyman G, Velez-García E, et al.: A phase III randomized study comparing cisplatin and fluorouracil as single agents and in combination for advanced squamous cell carcinoma of the head and neck. J. Clin. Oncol. 1992 Feb; 10(2): 257–263.\n\nClavel M, Vermorken JB, Cognetti F, et al.: Randomized comparison of cisplatin, methotrexate, bleomycin and vincristine (CABO) versus cisplatin and 5-fluorouracil (CF) versus cisplatin (C) in recurrent or metastatic squamous cell carcinoma of the head and neck. A phase III study of the EORTC Head and Neck Cancer Cooperative Group. Ann. Oncol. 1994 Jul; 5(6): 521–526. PubMed Abstract \| Publisher Full Text\n\nGibson MK, Li Y, Murphy B, et al.: Randomized phase III evaluation of cisplatin plus fluorouracil versus cisplatin plus paclitaxel in advanced head and neck cancer (E1395): an intergroup trial of the Eastern Cooperative Oncology Group. J. Clin. Oncol. 2005 May 20; 23(15): 3562–3567. PubMed Abstract \| Publisher Full Text\n\nSchöffski P, Catimel G, Planting AS, et al.: Docetaxel and cisplatin: an active regimen in patients with locally advanced, recurrent or metastatic squamous cell carcinoma of the head and neck. Results of a phase II study of the EORTC Early Clinical Studies Group. Ann. Oncol. 1999 Jan; 10(1): 119–122. Publisher Full Text\n\nGlisson BS, Murphy BA, Frenette G, et al.: Phase II Trial of docetaxel and cisplatin combination chemotherapy in patients with squamous cell carcinoma of the head and neck. J. Clin. Oncol. 2002 Mar 15; 20(6): 1593–1599. Publisher Full Text\n\nSpecht L, Larsen SK, Hansen HS: Phase II study of docetaxel and cisplatin in patients with recurrent or disseminated squamous-cell carcinoma of the head and neck. Ann. Oncol. 2000 Jul; 11(7): 845–849. Publisher Full Text\n\nBaur M, Kienzer H-R, Schweiger J, et al.: Docetaxel/cisplatin as first-line chemotherapy in patients with head and neck carcinoma: a phase II trial. Cancer. 2002 Jun 1; 94(11): 2953–2958. PubMed Abstract \| Publisher Full Text\n\nStathopoulos GP, Rigatos S, Papakostas P, et al.: Effectiveness of paclitaxel and carboplatin combination in heavily pretreated patients with head and neck cancers. Eur. J. Cancer. 1997 Oct; 33(11): 1780–1783. PubMed Abstract \| Publisher Full Text\n\nClark JI, Hofmeister C, Choudhury A, et al.: Phase II evaluation of paclitaxel in combination with carboplatin in advanced head and neck carcinoma. Cancer. 2001 Nov 1; 92(9): 2334–2340. Publisher Full Text\n\nSamlowski WE, Moon J, Kuebler JP, et al.: Evaluation of the combination of docetaxel/carboplatin in patients with metastatic or recurrent squamous cell carcinoma of the head and neck (SCCHN): a Southwest Oncology Group Phase II study. Cancer Investig. 2007 May; 25(3): 182–188. PubMed Abstract \| Publisher Full Text\n\nBrockstein BE, Vokes EE: Treatment of metastatic and recurrent head and neck cancer - UpToDate. UpToDate.2020 [cited 2020 Dec 9]. Reference Source\n\nLynggaard CD, Therkildsen MH, Kristensen CA, et al.: The EXTREME regimen for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC): treatment outcome in a single institution cohort. Acta Oncol. 2015 Jul; 54(7): 1071–1075. PubMed Abstract \| Publisher Full Text\n\nVermorken JB, Mesia R, Rivera F, et al.: Platinum-Based Chemotherapy plus Cetuximab in Head and Neck Cancer. N. Engl. J. Med. 2008 Sep 11; 359(11): 1116–1127. Publisher Full Text\n\nGuigay J, Fayette J, Mesia R, et al.: TPExtreme randomized trial: TPEx versus Extreme regimen in 1st line recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). JCO. 2019 May 20; 37(15_suppl): 6002–6002. Publisher Full Text\n\nSano D, Fujisawa T, Tokuhisa M, et al.: Real-world Treatment Outcomes of the EXTREME Regimen as First-line Therapy for Recurrent/Metastatic Squamous Cell Carcinoma of the Head and Neck: A Multi-center Retrospective Cohort Study in Japan. Anticancer Res. 2019 Dec 1; 39(12): 6819–6827. PubMed Abstract \| Publisher Full Text\n\nGuigay J, Fayette J, Dillies AF, et al.: Cetuximab, docetaxel, and cisplatin as first-line treatment in patients with recurrent or metastatic head and neck squamous cell carcinoma: a multicenter, phase II GORTEC study. Ann. Oncol. 2015 Sep 1; 26(9): 1941–1947. PubMed Abstract \| Publisher Full Text\n\nGuigay J, Tahara M, Licitra L, et al.: The Evolving Role of Taxanes in Combination With Cetuximab for the Treatment of Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck: Evidence, Advantages, and Future Directions. Front. Oncol. 2019 Aug 21 [cited 2020 Dec 4]; 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriesland S, Tsakonas G, Kristensen C, et al.: Randomised phase II study with cetuximab in combination with 5-FU and cisplatin or carboplatin versus cetuximab in combination with paclitaxel and carboplatin for treatment of patients with relapsed or metastatic squamous cell carcinoma of the head and neck (CETMET trial). JCO. 2018 May 20; 36(15_suppl): 6032–6032. Publisher Full Text\n\nSacco AG, Cohen EE: Current Treatment Options for Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma. JCO. 2015 Sep 8; 33(29): 3305–3313. Publisher Full Text\n\nLau A, Yang W, Li K-Y, et al.: Systemic Therapy in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma - A Systematic Review and Meta-Analysis. Crit. Rev. Oncol. Hematol. 2020 Sep 1; 153: 102984. PubMed Abstract \| Publisher Full Text\n\nBlasco MA, Svider PF, Raza SN, et al.: Systemic therapy for head and neck squamous cell carcinoma: Historical perspectives and recent breakthroughs. Laryngoscope. 2017; 127(11): 2565–2569. PubMed Abstract \| Publisher Full Text\n\nBurtness B, Harrington KJ, Greil R, et al.: Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet. 2019 23; 394(10212): 1915–1928. Publisher Full Text\n\nSpecenier P, Vermorken JB: Cetuximab: its unique place in head and neck cancer treatment. Biologics. 2013; 7: 77–90. PubMed Abstract \| Publisher Full Text\n\nSzturz P, Vermorken JB: Further clinical interpretation and implications of KEYNOTE-048 findings. Lancet. 2020 Aug 8; 396(10248): 379–380. PubMed Abstract \| Publisher Full Text\n\nNandakumar A, Nandakumar A: Survival in Head and Neck Cancers - Results of A Multi- Institution Study. Asian Pac. J. Cancer Prev. 2016; 17(4): 1745–1754. PubMed Abstract \| Publisher Full Text\n\nEisenhauer EA, Therasse P, Bogaerts J, et al.: New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer. 2009 Jan; 45(2): 228–247. PubMed Abstract \| Publisher Full Text\n\nCTCAE: Common Terminology Criteria for Adverse Events (CTCAE)\|Protocol Development\|CTEP.2018 [cited 2019 Oct 23]. Reference Source\n\nTahara M, Kiyota N, Yokota T, et al.: Phase II trial of combination treatment with paclitaxel, carboplatin and cetuximab (PCE) as first-line treatment in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (CSPOR-HN02). Ann. Oncol. 2018 Apr 1; 29(4): 1004–1009. PubMed Abstract \| Publisher Full Text\n\nAdkins D, Ley J, Atiq O, et al.: Multicenter Phase 2 Trial of Cis/Carboplatin, nAb-Paclitaxel, and CeTUXimab (CACTUX) as First-Line Therapy for Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 2018 Apr 1; 100(5): 1311–1312. Publisher Full Text\n\nPêtre A, Dalban C, Karabajakian A, et al.: Carboplatin in combination with weekly Paclitaxel as first-line therapy in patients with recurrent/metastatic head and neck squamous cell carcinoma unfit to EXTREME schedule. Oncotarget. 2018 Apr 24; 9(31): 22038–22046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzturz P, Cristina V, Herrera Gómez RG, et al.: Cisplatin Eligibility Issues and Alternative Regimens in Locoregionally Advanced Head and Neck Cancer: Recommendations for Clinical Practice. Front. Oncol. 2019 [cited 2019 Oct 20]; 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgarwala V, Chandrakanth MV, Mondal P, et al.: TP Ex for analysis- Weekly paclitaxel baseline data- Revised.xlsx. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "263664", "date": "26 Apr 2024", "name": "Ye Guo", "expertise": [ "Reviewer Expertise head and neck cancer", "anti-cancer phase 1 clinical trial" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a retrospective study looking at the efficacy of PCC regimen in 1st line and nivolumab or oral metronomic chemotherapy in 2nd line in R/M HNSCC. Comments:\nAs a retrospective study for R/M HNSCC, it is surprising that the author claims that “Written informed consent was obtained from each patient”. The author needs to clarify how to define the PFS and OS either in 1st and 2nd line. The main objective of this study is to analyze the efficacy of PCC regimen in 1st line, but the author didn’t report the OS data for the total 54 patients enrolled. The author reported the OS for 29 patients who received the salvage treatment in 2nd line, but exclusion of 4 patients who managed by BSC may bias the results in this setting. According to the conclusion of “The addition of 2L nivolumab on progression further improves the outcomes”, but the author didn’t report the OS for patients who were treated with PCC regimen followed by nivolumab salvage, although the OS for the salvage treatment was high numerically and even much better as compared to that reported in CheckMate-141 study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "11577", "date": "15 Jul 2024", "name": "Vivek Agarwala", "role": "Author Response", "response": "Dear Reviewer, Thank you for your thorough review and for pointing out the limitations of our retrospective analysis. We genuinely appreciate your effort in assessing our study and providing constructive feedback. This is a retrospective study looking at the efficacy of the PCC regimen in the first line and nivolumab or oral metronomic chemotherapy in the second line in R/M HNSCC. Comments: As a retrospective study for R/M HNSCC, it is surprising that the author claims that “Written informed consent was obtained from each patient”. Response: In our study, we encountered some challenges with patient eligibility and willingness to undergo second-line therapy in a potentially fatal disease like head and neck cancer. As a result, four patients in our sample were excluded from receiving further treatment due to their unfitness or lack of willingness. The author needs to clarify how to define the PFS and OS either in 1st and 2nd line. Response: However, it is important to note that we included these patients in calculating the overall survival rates for the entire population. Despite the limitations of our study design, we found a significant difference in overall survival between those who received nivolumab as a subsequent line of treatment and those who received either oral metronomic therapy or best supportive care. OS was defined as the time from the date of diagnosis till the date of death due to any cause. Progression free survival (PFS) was defined as the time from first administration of PCC regimen alone to the first radiological confirmation of disease progression The main objective of this study is to analyze the efficacy of PCC regimen in 1st line, but the author didn’t report the OS data for the total 54 patients enrolled. Response: We apologize for any confusion or oversight caused by not explicitly mentioning this limitation in our initial publication. We genuinely value your feedback and suggestions for future research. The author reported the OS for 29 patients who received the salvage treatment in 2nd line, but exclusion of 4 patients who managed by BSC may bias the results in this setting. According to the conclusion of “The addition of 2L nivolumab on progression further improves the outcomes”, but the author didn’t report the OS for patients who were treated with PCC regimen followed by nivolumab salvage, although the OS for the salvage treatment was high numerically and even much better as compared to that reported in CheckMate-141 study. Response: In light of your comments, we agree that conducting a prospective study would be beneficial to further investigate the efficacy and potential differences between nivolumab and oral metronomic therapy or best supportive care in the treatment of head and neck cancer. By considering your suggestions, we aim to enhance the robustness and reliability of our findings. Once again, we sincerely thank you for taking the time to review our study and for your valuable input. Your expertise and insights contribute significantly to our efforts to improve the quality and credibility of our research." } ] }, { "id": "227529", "date": "14 May 2024", "name": "Santiago Cabezas-Camarero", "expertise": [ "Reviewer Expertise Head and neck oncology", "CNS oncology", "G-U oncology", "Genetic counseling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChandrakanth et al, report an interesting real-world study on the efficacy and safety of the weekly PCC regimen (paclitaxel, carboplatin, cetuximab) followed at progression by either the anti-PD1 nivolumab or oral metronomic therapy (OMT: celecoxib, erlotinib, methotrexate). The data on the sequence of PCC followed by nivolumab achieved a notable OS of 20.6 months (N=19) compared to 7 months in OMT (N=10), and 15.1 months in the entire cohort.\nHowever they should address several issues:\n\nMajor remark: the authors should discuss more extensively on the sequence of platinum-based chemo followed by ICI such as the data reported in Checkmate-141, the OS data of EXTREME/TPEX followed by ICI in TPEXTREME, OS data of the control arm of Kestrel study (Extreme followed by ICI compared to other therapies or no treatment), OS data of the control arm of Checkmate-651 (Extreme followed by ICI in 46% of patients). In addition, in the RWE study TTCC-2019-02 patients treatment with ERBITAX followed by ICI achieved a median OS of nearly 30 months (Front Oncol 2023 Aug 1:13:1226939).\n\nOther remarks: -Discussion: The authors state that \"the TPEXTREME regimen did not look at PFS and ORR\" --> This is completely incorrect and should be erased and rediscussed.\nThe authors state that in KN-048 authors had mainly non-oral primaries and HPV related etiology --> This is not accurate since KN-048 was a global study and the population with oral cavity cancers was nearly 30% and that with HPV-positive oropharyngeal was 20-30%. The authors should avoid such statements.\n\"Indian patients have a far higher disease burden than that of the Western population\" --> The authors should add a reference to justify this.\nDiscussion, Final paragraph: \"...our study has contributed immensely...\" --> They should avoid autolaudatory statements...\n-Table 4: the authors should detail the combination of each regimen. The authors should include other first-line cetuximab-based regimens such as ERBITAX (i.e. Hitt et al Ann Oncol 2012). The reference for the TPEXTREME study (Ref. 18) is outdated --> They should include the definitive journal publication and include the data of ORR and PFS.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "13184", "date": "23 Jan 2025", "name": "Vivek Agarwala", "role": "Author Response", "response": "Dear Reviewer, Thank you for your thorough review and for pointing out the limitations of our retrospective analysis. We genuinely appreciate your effort in assessing our study and providing constructive feedback. Comments: 1. The authors should discuss more extensively on the sequence of platinum-based chemo followed by ICI such as the data reported in Checkmate-141, the OS data of EXTREME/TPEX followed by ICI in TPEXTREME, OS data of the control arm of Kestrel study (Extreme followed by ICI compared to other therapies or no treatment), OS data of the control arm of Checkmate-651 (Extreme followed by ICI in 46% of patients). In addition, in the RWE study TTCC-2019-02 patients treatment with ERBITAX followed by ICI achieved a median OS of nearly 30 months (Front Oncol 2023 Aug 1:13:1226939). Response: When this manuscript was initially written & submitted for review at that time, data from Kestrel study, Checkmate-651 & TTCC-2019-02 was not presented. We have discussed these trials in the discussion of the updated manuscript. 2. The authors state that \"the TPEXTREME regimen did not look at PFS and ORR\" --> This is completely incorrect and should be erased and re-discussed. Response: Thanks for sharing that, it has been removed from manuscript. ORR & PFS data of TPExtreme study included. 3. The authors state that in KN-048 authors had mainly non-oral primaries and HPV related etiology --> This is not accurate since KN-048 was a global study and the population with oral cavity cancers was nearly 30% and that with HPV-positive oropharyngeal was 20-30%. The authors should avoid such statements. Response: Those statements updated. 4. \"Indian patients have a far higher disease burden than that of the Western population\" --> The authors should add a reference to justify this. Response: Reference added. 5. Discussion, Final paragraph: \"...our study has contributed immensely...\" --> They should avoid autolaudatory statements. Response: Rephrased by deleting immensely. 6. Table 4: the authors should detail the combination of each regimen. The authors should include other first-line cetuximab-based regimens such as ERBITAX (i.e. Hitt et al Ann Oncol 2012). The reference for the TPEXTREME study (Ref. 18) is outdated --> They should include the definitive journal publication and include the data of ORR and PFS. Response: Table & reference updated." } ] } ]	1	https://f1000research.com/articles/12-802
https://f1000research.com/articles/13-280/v1	16 Apr 24	{ "type": "Research Article", "title": "Revisiting Mac-2-Binding Protein Glycosylation Isomer (M2BPGi) for Diagnosing High-Risk Liver Fibrosis: A Stepwise Diagnostic Analysis", "authors": [ "Muhammad Begawan Bestari", "Haryono Haryono", "Muhammad Palar Wijaya", "Dolvy Girawan", "Nenny Agustanti", "Eka Surya Nugraha", "Haryono Haryono", "Muhammad Palar Wijaya", "Dolvy Girawan", "Nenny Agustanti", "Eka Surya Nugraha" ], "abstract": "Abstract\nBackground The level of liver fibrosis is the basis for the treatment of chronic hepatitis B (CHB), and it is necessary to adapt non-invasive liver fibrosis modalities. We aimed to investigate the use of M2BPGi as a single or combined diagnostic modality for liver fibrosis in CHB patients through a stepwise diagnostic analysis.\n\nMethods Cross-sectional data were taken from patients between October 2021 and August 2022. Demographic data, blood profile, liver function, and liver stiffness were measured in CHB patients over 18 years old, willing to take part in the research, and had complete data. APRI, FIB-4, and AAR were calculated using the well-known formulas. Serum M2BPGi-levels were converted into a cut-off index (COI). The patients were divided into low-risk (LR) and high-risk fibrosis (HR) groups. A cut-off for each predictor variable to differentiate between the LR and HR groups was determined. The obtained cut-off was assessed for its association with the grouping of liver elastography results. Models to diagnose the liver stiffness measurement (LSM) ≥8 kPa were created and compared through multivariate and ROC analyses.\n\nResults The number of patients that met the inclusion and exclusion criteria was 143 (HR = 65, LR = 78). The cut-off for diagnosing LSM ≥8kPa was 0.311, 0.742, 0.635, and 1.434 for APRI, FIB-4, AAR, and M2BPGi, respectively. This cut-off was significantly associated with the results of the HR and LR groupings. A multivariate analysis found that FIB4, AAR, and M2BPGi added significantly to the model. Statistically, the most optimal use of M2BPGi was combined with FIB-4, with an AUC of 0.835.\n\nConclusions The optimal cut-off of M2BPGi for diagnosing high-risk liver fibrosis in this study was 1.434. M2BPGi should be used with FIB-4 as a diagnostic tool for diagnosing liver fibrosis, especially in the absence of a liver biopsy or elastography.", "keywords": [ "M2BPGi", "chronic hepatitis B", "fibrosis", "diagnostic", "APRI", "FIB-4", "AAR" ], "content": "Introduction\n\nGlobally, an estimated 296 million people, with 18 million in Southeast Asia, are projected to have a CHB infection by the World Health Organization (WHO). The annual rate of new infections is about 1.5 million. Hepatocellular carcinoma (HCC) and liver fibrosis caused by hepatitis B were responsible for 820,000 deaths in 2019.1 In determining the severity of fibrosis or inflammation in the liver, a liver biopsy is the primary option, but it is an invasive procedure. The American Association for the Study of Liver Diseases (AASLD) has suggested several non-invasive techniques.2\n\nMac-2-binding protein (M2BP) is a glycoprotein that, when changes are made to its N-glycan residue, forms M2BPGi. M2BPGi is produced by hepatic stellate cells (HSCs) and it induces profibrotic cytokine expression in Kupffer cells (KCs), namely Mac-2. Subsequently, Mac-2 activates HSCs and cause fibrogenesis.3,4 M2BPGi has been widely used to predict liver fibrosis and cirrhosis in different chronic liver diseases.4–17 In several previous studies, M2BPGi helped to diagnose liver fibrosis in a CHB population5,10 and it could be used as a single predictor variable to diagnose liver fibrosis grade.11,12 This marker could also complement and be used with other modalities.13,14 The most accurate non-invasive methods to assess fibrosis are liver stiffness measurements (elastography), followed by several scoring methods such as the AST-to-platelet ratio index (APRI), the fibrosis index based on four factors (FIB-4), and the AST-to-ALT ratio (AAR).2,18–20\n\nAdapting non-invasive liver fibrosis modalities to each type of chronic liver disease and each region is necessary due to the heterogeneity of outcomes. A stepwise diagnostic analysis has yet to be conducted to determine whether M2BPGi should be utilized alone or in conjunction with modalities to assess liver fibrosis. Thus, we aimed to investigate the use of M2BPGi as a single or combined diagnostic modality for liver fibrosis in CHB patients through a stepwise diagnostic analysis.\n\n\nMethods\n\nWe obtained ethical approval from the Research Ethics Committee of Dr. Hasan Sadikin General Hospital Bandung (LB.02.01/X.6.5/299/2021) in order to protect the rights and welfare of research subjects, and to guarantee the study to be conducted according to ethical, legal, social implications, and other applicable regulations. This was a cross-sectional study; the subjects of this study were patients from the Gastroenterohepatology outpatient clinic, Hasan Sadikin General Hospital, Indonesia, between October 2021 and August 2022. All patients were older than 18 years and were positive for serum hepatitis B surface antigen (HBsAg) for at least six months. The criteria for exclusion were as follows: 1) acute hepatitis; 2) acute exacerbation of chronic hepatitis; 3) hepatitis C; 4) autoimmune liver disease; 5) hepatitis B co-infection with hepatitis C or HIV; 6) co-morbidities (type 2 diabetes mellitus, heart disease, chronic kidney disease, pulmonary tuberculosis, or cancer); 7) patients with a history of alcohol use (>20 grams of alcohol per day); 8) pregnant or breastfeeding woman; 9) body mass index (BMI) >27 kg/m2; 10) hemoglobin <5 g/dL; and 11) pulmonary fibrosis, chronic pancreatitis, liver cancer, or pancreatic cancer.\n\nThere are several ways to determine the optimum sample size for a binary logistic regression analysis. First, using the rule of thumb method with N = number of independent variables multiplied by 10-50, the value for our sample size was between 30 and 150. Another method is by including the prevalence correction factor with the formula: N = 10 k/p, where k is the number of independent variables and p is the prevalence correction factor.21 In our study subjects, the prevalence was 45%; thus, the number of efficient samples is 66.67 = 67 patients. Our study was conducted on 143 subjects.\n\nCHB patients who met the inclusion and exclusion criteria received information about the study. After obtaining written (informed) consent, their demographic data were collected. The research subjects underwent supporting examinations of liver elastography and routine laboratory investigations, including measurements of CBC, AST, ALT, PT, INR, and M2BPGi serum levels. All laboratory examinations were carried out in the clinical pathology laboratory of Hasan Sadikin General Hospital. The formulas used to calculate the non-invasive liver fibrosis scores are as follows22,23:\n\nTransient elastography (TE) is a non-invasive examination to determine the level of liver fibrosis, with results given in the form of an LSM (in kPa). This examination was performed using the FibroScan® 502 Series F00734 (Echosens, Paris, France) with the M or XL probe. Liver stiffness was expressed as the median value of more than ten valid examinations. The value of LSM can be trusted if the success rate is greater than 60% and the interquartile range (IQR) ratio to the median liver stiffness is below 30%. This examination was performed by a gastroenterohepatologist at Hasan Sadikin General Hospital.\n\nSerum M2BPGi levels were measured using the HISCL M2BPGi reagent kit (Sysmex, Hyogo, Japan, Catalogue number: CB090850) (Supplier: PT. Saba Indomedika) and an automatic immune analyzer HISCL 800 (Sysmex, Hyogo). In total, from the M2BPGi reagent kit, 50 μL of R1 reagent, 30 μL of R2 reagent, 600-4200 μL of washing solution, 100 μL of R3 reagent, 50 μL of R4 reagent, and 100 μL of R5 reagent, were used. The results of the M2BPGi serum level were expressed as a cut-off index (COI). The COI was calculated using the following formula24:\n\nFirst, a normality test was conducted to determine the subsequent statistical analysis procedure. The patient characteristic data are presented in Table 1; those with a normal distribution are expressed as the mean and standard deviation, while those that are not normally distributed are presented as the median and minimum–maximum values. The results of the transient elastography were used to classify the subjects into two groups: high-risk fibrosis (HR) (LSM ≥8 kPa) and low-risk fibrosis (LR) (LSM < 8 kPa).25\n\n Value is given as a proportion.\n\n† Values are the medians with ranges in parentheses.\n\n‡ Values are the means with standard deviations in parentheses.\n\nEach predictor variable underwent an ROC analysis using SPSS version 20 (IBM Corp. 2011. Armonk, NY, USA, RRID:SCR_00286) to develop cut-off values based on Youden’s index to balance sensitivity and specificity in diagnosing high-risk fibrosis. STATA 17 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC; RRID: SCR_012763) was used for the stepwise diagnostic analysis. Each predictor variable was grouped based on their cut-off value. The association between each grouped predictor variable and the HR and LR groupings was assessed using a chi-square analysis. Diagnostic models were created and subjected to stepwise logistic regression. The receiver operating characteristic (ROC) analysis determined each model’s accuracy. Then, ROC analysis comparisons were carried out between each model to evaluate whether there was a significant difference in adding predictors. A two-tailed p < 0.05 was considered statistically significant.\n\n\nResults\n\nThe total number of CHB patients at Hasan Sadikin General Hospital during the research period was 157. All patients were entered into the Hasan Sadikin Chronic Hepatitis B Registry. After all examinations and data collection, 14 patients were excluded because of incomplete data. The number of patients who met the inclusion and exclusion criteria was 143. The patients’ baseline characteristics are summarized in Table 1. Based on the transient elastography results, the patients were split into two groups: high-risk fibrosis (HR) (n = 65) and low-risk fibrosis (LR) (n = 78) groups. The flowchart for the selection of the participants is shown in Figure 1.\n\nEach predictor underwent an ROC analysis to obtain the optimal cut-off point. It was found that the best cutoff values for M2BPGi, APRI, FIB-4, and AAR were 1.434, 0.311, 0.742, and 0.635, respectively. The results of the bivariate analysis between the predictor variables for the HR and LR groups are shown in Table 2. Based on the liver elastography results, M2BPGi, APRI, FIB-4, and AAR were significantly associated with the HR and LR groupings.\n\n* p-value from the chi-square test. LSM: liver stiffness measurement; M2BPGi Mac-2-binding protein glycosylation isomer; APRI: AST-to-platelet ratio index; FIB4: fibrosis index based on 4 factors; AAR: AST-to-ALT ratio.\n\nThrough a Spearman analysis, APRI with FIB-4 had the strongest correlation, with a correlation coefficient of 0.78. Based on the correlation coefficient data, it was decided to exclude APRI from the diagnostic model to avoid violating the multicollinearity rule. The models used different combinations of M2BPGi, FIB-4, and AAR. Model 1 consisted of M2BPGi only, followed by the addition of other predictors one by one to produce Model 2, which consisted of M2BPGi and AAR; Model 3, which consisted of M2BPGi and FIB-4; and Model 4, which consisted of M2BPGi, FIB-4, and AAR. All variables were analyzed to assess the association. The results of the multivariate analysis are shown in Table 3. All models were statistically significant (p-value < 0.001) compared to the intercept-only model and had a good pseudo-R2 fit at 0.2, 0.23, 0.31, and 0.33 for Models 1, 2, 3, and 4, respectively.\n\nThe ROC analysis’s area under the curve (AUC) evaluates each model’s accuracy (Table 4). The model utilizing M2BPGi with the best accuracy was in combination with FIB-4 and AAR. The abilities of each model were compared, and the results are shown in Table 5.\n\n* Statistically significant difference.\n\n\nDiscussion\n\nNon-invasive methods for assessing liver fibrosis are currently being developed. It has been demonstrated that M2BPGi is a good biomarker for evaluating liver fibrosis.4–17 A stepwise diagnostic analysis has not yet been conducted to determine the value of M2BPGi in assessing liver fibrosis. Here, we aimed to investigate the use of M2BPGi as a single or combined diagnostic modality for liver fibrosis in CHB patients through a stepwise diagnostic analysis.\n\nCurrently, a liver biopsy is the gold standard for assessing liver inflammation and fibrosis. However, biopsy is an invasive procedure and has several risks, such as bleeding, hematoma, and mild discomfort to severe pain; hence, it is not suitable for routine use.26 There are various non-invasive methods to assess liver fibrosis. Liver elastography is the primary alternative for assessing liver fibrosis. To determine liver stiffness in this study, we used the FibroScan® tool and determined the fibrosis class based on the EASL recommendations in FibroScan®. In the outside liver clinic settings, the results of LSM are divided into ≥8 kPa for high-risk fibrosis and <8 kPa for low-risk.25 Operator skills and experience, the selection of appropriate probes, and special conditions such as obesity are challenges in applying the liver elastography method.\n\nThe ROC curve analysis obtained a new cut-off for M2BPGi, APRI, FIB-4, and AAR (Table 2). This optimal cut-off is not too different from those of Zou’s study14 for diagnosing METAVIR grade ≥F2 with an APRI cut-off of 0.51, a FIB-4 cut-off of 0.92, and an AAR cut-off of 0.55. In a study to distinguish LSM ≥7 kPa in Vietnam, cut-offs of 0.5 and 1.8 were obtained for APRI and FIB-4, respectively.11 In patients with liver elastography results ≥9 kPa in Egypt, the cut-offs were at 0.256, 0.74, and 0.8 for APRI, FIB-4, and AAR, respectively.27 For predicting Knodell histologic activity index (HAI) ≥F2 results, APRI and FIB-4 had the best cut-offs at 0.9 and 0.35, respectively.10 The research regarding the ability of non-invasive liver fibrosis modalities can be broadly divided into two areas, using liver biopsy or TE as the comparison. We are among those who used TE results as the gold standard. While TE was rarely used, Bui et al. found an M2BPGi cut-off of 0.79 for diagnosing LSM ≥7 kPa.11 The cut-off of M2BPGi that we obtained to diagnose LSM ≥8 kPa was 1.434. Our cut-off is quite close to the previous cut-off for diagnosing significant liver fibrosis using biopsy as the gold standard by Yeh et al.13 and Ishii et al.5 at 1.345 and 1.4, respectively.\n\nOur M2BPGi cut-off is greater than 1, which we suspect is due to aging. There was a significant difference between the ages of the HR and LR groups in our study. In several previous studies, the cut-off for M2BPGi was around COI 1.5,9,10,12,14 Cheng et al. found that aging increases M2BPGi levels in healthy patients.17 This finding may explain why our cut-off results were more than one. However, the effects aging on M2BPGi levels require further research. Based on the cut-off found, the four predictor variables were divided into categorical data; all predictors were associated with the categorical classification of liver elastography with a cut-off of 8 kPa (Table 2).\n\nThe highest bivariate correlation analysis results were found between APRI and FIB-4; this was based on the fact that both indices consist of AST and platelet counts as the primary variables. Four models were developed involving M2BPGi, FIB-4, and AAR to assess the performance of M2BPGi on its own. M2BPGi, FIB-4, and AAR (Model 4) were able to predict the HR group. Patients with any result equal to or more than the M2BPGi, FIB-4, and AAR cut-offs will result in a probability of 6.476, 6.873, and 2.685, respectively, for classification into the HR group. If used alone, each COI M2BPGi value ≥1.434 will produce a probability of 11.562 (Table 4).\n\nModel 4 had the best diagnostic ability with an AUC of 0.852 (Table 4). The use of M2BPGi as a single modality (Model 1) in diagnosing high-risk liver fibrosis was quite good, with an AUC of 0.733. In diagnosing liver biopsy at ≥F2, Yeh et al.13 and Zou et al.14 obtained an AUC for M2BPGi of 0.78 and 0.753, respectively. Bui et al. found an AUC of 0.77 for diagnosing LSM ≥7 kPa.11 Ichikawa et al., in determining F≥2 based on the revised Inuyama classification, found that M2BPGi had an AUC of 0.713.9 In the group of patients with treatment-naïve CHB to diagnose portal fibrosis without septal involvement (F≥2), an AUC of 0.77 was obtained by Ishii et al.5\n\nThere were significant differences in the diagnostic abilities of the models (p = 0.0019); a post hoc analysis was performed to determine whether the addition of a modality was statistically significant (Table 5). In Model 2, M2BPGi was coupled with AAR, which increased the diagnostic capability compared to the M2BPGi-only model by around 4.9%. Model 3, which consisted of M2BPGi and FIB-4, was statistically the best model, with an AUC of 0.835. The addition of FIB-4 increased the AUC to 10.2%. Adding AAR to Model 3 to form Model 4 increased the diagnostic capability by 1.7% but this was not statistically significant. Therefore, Model 3 was the most efficient diagnostic model. M2BPGi can be used efficiently, and its application should be combined with FIB-4 to diagnose high-risk liver fibrosis.\n\nIn some earlier studies, the use of M2BPGi combined with other variables was proposed, as was performed by Yeh et al.13 and Zou et al.14 Yeh et al. supported using M2BPGi in models involving age and platelet counts to increase the specificity in the prediction of advanced fibrosis.13 Zou et al. suggested measuring M2BPGi levels as a complementary method for liver biopsies and elastography.14 However, both studies showed that the AUC value of M2BPGi was always superior to other scoring methods.13,14 Bui et al. found that M2BPGi and APRI had the same AUC value (0.77) as a single indicator. However, in combining M2BPGi with other modalities, they only formed a single model to predict significant fibrosis using M2BPGi and APRI. By adding APRI to M2BPGi, the accuracy in detecting LSM ≥7 kPa was increased. Based on the high coefficient correlation between M2BPGi and liver elastography results, the paper stated that M2BPGi could be used as an alternative liver fibrosis test in CHB patients, especially in settings with limited resources.11 Mak et al. performed an ROC analysis and created two predictive models for F3/F4 biopsy results. M2BPGi always produced statistically significant correlation in both models, while APRI, FIB-4, and AAR did not. They stated that M2BPGi was a potential marker for easily diagnosing F3/F4 without the need for a liver biopsy.12\n\nThis study of serum M2BPGi levels in CHB patients aimed to aid its diagnostic application outside of liver clinic settings. The use of M2BPGi levels as part of a non-invasive method for diagnosing liver fibrosis outcomes based on liver elastography values was compared with several scoring methods. In our study, M2BPGi showed good diagnostic performance when used alone. However, our stepwise diagnostic analysis found that M2BPGi had a better result in diagnosing liver fibrosis when combined with FIB-4.\n\nSince this is the first comprehensive statistical analysis performed on M2BPGi utilization, future studies should examine the use of serum M2BPGi levels by applying the stepwise diagnostic analysis method. In conclusion, after considering all the statistical comparisons and the stepwise diagnostic analysis, we believe that M2BPGi should be used with FIB-4 as a diagnostic tool for liver fibrosis, especially in the absence of liver biopsies or elastography.\n\nThe Research Ethics Committee of Dr. Hasan Sadikin General Hospital Bandung granted permission on 20 October 2021 to conduct this research (LB.02.01/X.6.5/299/2021). After obtaining written (informed) consent, their demographic data were collected.", "appendix": "Data availability\n\nFigshare: CHB for stepwise M2BPGi, https://doi.org/10.6084/m9.figshare.24971649.v1. 28\n\nThis project contains the following underlying data:\n\n• CHB for stepwise M2BPGi.sav (the raw data for the study)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Participant information sheets, https://doi.org/10.6084/m9.figshare.25383106.v1. 29\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Informed consent form for sample, https://doi.org/10.6084/m9.figshare.25383157.v1. 30\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nSTARD checklist for ‘Revisiting Mac-2-Binding Protein Glycosylation Isomer (M2BPGi) for Diagnosing High-Risk Liver Fibrosis: A Stepwise Diagnostic Analysis’. https://doi.org/10.6084/m9.figshare.25383313.v1\n\n\nAcknowledgements\n\nThe authors sincerely thank the dedicated staff at the Gastroenterohepatology Outpatient Clinic and the Clinical Pathology Laboratory of Hasan Sadikin General Hospital for their invaluable contributions and unwavering support in this research journey.\n\n\nReferences\n\nWHO: Hepatitis B fact sheet.2022 [cited 2021 Apr 10]. Reference Source\n\nTerrault NA, Lok ASF, McMahon BJ, et al.: Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology. 2018; 67: 1560–1599. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBekki Y, Yoshizumi T, Shimoda S, et al.: Hepatic stellate cells secreting WFA+-M2BP: Its role in biological interactions with Kupffer cells. J. Gastroenterol. Hepatol. 2017; 32: 1387–1393. PubMed Abstract \| Publisher Full Text\n\nShirabe K, Bekki Y, Gantumur D, et al.: Mac-2 binding protein glycan isomer (M2BPGi) is a new serum biomarker for assessing liver fibrosis: more than a biomarker of liver fibrosis. J. Gastroenterol. 2018; 53: 819–826. PubMed Abstract \| Publisher Full Text\n\nIshii A, Nishikawa H, Enomoto H, et al.: Clinical implications of serum Wisteria floribunda agglutinin-positive Mac-2-binding protein in treatment-naïve chronic hepatitis B. Hepatol. Res. 2017; 47: 204–215. PubMed Abstract \| Publisher Full Text\n\nSulaiman AS, Hasan I, Lesmana CRA, et al.: Diagnostic Performance of Mac-2-Binding Protein Glycosylation Isomer (M2BPGi), compared to Transient Elastography to Assess Liver Stiffness in Treatment Naïve Chronic Hepatitis C Patients. Acta Med. Indones. 2022; 54: 567–573. PubMed Abstract\n\nXu H, Kong W, Liu L, et al.: Accuracy of M2BPGi, compared with Fibro Scan®, in analysis of liver fibrosis in patients with hepatitis C. BMC Gastroenterol. 2017; 17: 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaleh SA, Salama MM, Alhusseini MM, et al.: M2BPGi for assessing liver fibrosis in patients with hepatitis C treated with direct-acting antivirals. World J. Gastroenterol. 2020; 26: 2864–2876. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIchikawa Y, Joshita S, Umemura T, et al.: Serum Wisteria floribunda agglutinin-positive human Mac-2 binding protein may predict liver fibrosis and progression to hepatocellular carcinoma in patients with chronic hepatitis B virus infection. Hepatol. Res. 2017; 47: 226–233. PubMed Abstract \| Publisher Full Text\n\nJekarl DW, Choi H, Lee S, et al.: Diagnosis of liver fibrosis with wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA-M2BP) among chronic hepatitis B patients. Ann. Lab. Med. 2018; 38: 348–354. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBui HH, Nguyen STB, Phan ST, et al.: Evaluating M2BPGi as a Marker for Liver Fibrosis in Patients with Chronic Hepatitis B. Dig. Dis. Sci. 2023; 68: 4407–4417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMak LY, Wong DKH, Cheung KS, et al.: Role of serum M2BPGi levels on diagnosing significant liver fibrosis and cirrhosis in treated patients with chronic hepatitis B virus infection. Clin. Transl. Gastroenterol. 2018; 9: e163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYeh ML, Huang CF, Huang C, et al.: Wisteria floribunda agglutinin-positive Mac-2binding protein in the prediction of disease severity in chronic hepatitis B patients. PLoS One. 2019; 14: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou X, Zhu MY, Yu DM, et al.: Serum WFA+-M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection. Liver Int. 2017; 37: 35–44. PubMed Abstract \| Publisher Full Text\n\nHur M, Park M, Moon HW, et al.: Comparison of non-invasive clinical algorithms for liver fibrosis in patients with chronic hepatitis B to reduce the need for liver biopsy: Application of enhanced liver fibrosis and Mac-2 binding protein glycosylation isomer. Ann. Lab. Med. 2021; 42: 249–257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu J, Hu HH, Lee MH, et al.: Serum Levels of M2BPGi as Short-Term Predictors of Hepatocellular Carcinoma in Untreated Chronic Hepatitis B Patients. Sci. Rep. 2017; 7: 14310–14352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng Y, Wang C: Comparison of Mac-2 binding protein glycosylation isomer (M2BPGi) with AST to platelet ratio index (APRI), fibrosis 4 Score (FIB -4), and nonalcoholic fatty liver disease (NAFLD) fibrosis score (NFS) for NAFLD patients. Adv. Dig. Med. 2022; 10: 87–95. Publisher Full Text\n\nKumbara CIYK, Wibawa IDN, Somayana IG, et al.: Diagnostic performance of APRI, RPR, FIB-4, GPR, AAR, and NLR in assessing the degree of liver fibrosis in patients with chronic hepatitis B. Indones. J. Biomed. Sci. 2020; 14: 122–127. Publisher Full Text\n\nDing D, Li H, Liu P, et al.: FibroScan, aspartate aminotransferase and alanine aminotransferase ratio (AAR), aspartate aminotransferase to platelet ratio index (APRI), fibrosis index based on the 4 factor (FIB-4), and their combinations in the assessment of liver fibrosis in patients. Int. J. Clin. Exp. Med. 2015; 8: 20876–20882. PubMed Abstract\n\nSha FR, Pk MU, Abuelezz NZ, et al.: Investigating the Efficiency of APRI, FIB-4, AAR and AARPRI as Noninvasive Markers for Predicting Hepatic Fibrosis in Chronic Hepatitis B Patients in Bangladesh. Open Microbiol. J. 2019; 13: 34–40. Publisher Full Text\n\nPeduzi P, Concato J, Kemper E, et al.: Practical methods for public health practitioners. J. Clin. Epidemiol. 1996; 49: 1373–1379. PubMed Abstract \| Publisher Full Text\n\nWai CT, Greenson JK, Fontana RJ, et al.: A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology. 2003; 38: 518–526. PubMed Abstract \| Publisher Full Text\n\nSterling RK, Lissen E, Clumeck N, et al.: Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology. 2006; 43: 1317–1325. PubMed Abstract \| Publisher Full Text\n\nKuno A, Sato T, Shimazaki H, et al.: Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling. Proteomics Clin. Appl. 2013; 7: 642–647. PubMed Abstract \| Publisher Full Text\n\nBerzigotti A, Tsochatzis E, Boursier J, et al.: EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis – 2021 update. J. Hepatol. 2021; 75: 659–689. PubMed Abstract \| Publisher Full Text\n\nMaria Streba L, Florin E, Teodor C: Risks and Benefits of Liver Biopsy in Focal Liver Disease. Liver Biopsy - Indications, Procedures, Results. InTech; 2012. Publisher Full Text\n\nBadawi R, Soliman H, Ziada D, et al.: Serum Markers as a Predictor of Hepatic Fibrosis Compared to Fibroscan in chronic hepatitis B Infected Egyptian patients: A Cross-sectional Study. Open Biomark J. 2020; 10: 69–75. Publisher Full Text\n\nWijaya MP: CHB for stepwise M2BPGi. figshare. Dataset. 2024. Publisher Full Text\n\nWijaya MP: Participant information sheets. figshare. Journal contribution. 2024. Publisher Full Text\n\nWijaya MP: Informed consent form for sample. figshare. Journal contribution. 2024. Publisher Full Text" }	[ { "id": "273641", "date": "14 Jun 2024", "name": "Kemal Fariz Kalista", "expertise": [ "Reviewer Expertise Hepatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this is very good work. This study comparing M2BPGi with elastography and many scoring system to assess liver fibrosis like APRI, FIB-4 and AAR, so i think this is a strength of this study, because not many studies comparing M2BPGi with multiple scoring system like this study. I consider to add “patient with chronic hepatitis B” in study title, because population in this study only include patient with hepatitis B. In discussion, I also consider to elaborate further regarding why APRI cut off for diagnosing liver fibrosis (0.311) is lower than many previous studies. It would be appreciate if author more explain why APRI was excluded from the model, because I think APRI is quite easy way to do in daily practice. Regarding study title “stepwise diagnosis analysis” I would suggest the author to provide how to apply study result to real life practice, eg: when considering patient for further fibrosis assessment with elastography or liver biopsy or if the M2BPGi result < 1.4 and FIB-4 > 0.7 what author suggestion in this kind of situation. Minor revision maybe it would be better if in table 2, in variable APRI, FIB-4, AAR and M2BPGi row, add note which one is lower or higher than cut off level. I think this study is approved with reservation, need to add further revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "11991", "date": "12 Jul 2024", "name": "MUHAMMAD BEGAWAN BESTARI", "role": "Author Response", "response": "The F1000 Research Dear Editor and reviewer (Dr Kemal Fariz Kalista), Subject: Submission of Revised Manuscript I hope this comment finds you well. I am writing to express my sincere gratitude for the feedback provided by the reviewer on our manuscript, “Revisiting Mac-2-Binding Protein Glycosylation Isomer (M2BPGi) for Diagnosing High-Risk Liver Fibrosis: A Stepwise Diagnostic Analysis.” The insightful comments and suggestions have significantly enhanced the quality and clarity of our work. In response to the reviewer's feedback, we have carefully addressed each comment and made substantial revisions to improve the overall quality of the manuscript. The critical revisions include: Clarification of Title: We have added information to the title to better represent our manuscript's contents. Clarification of Results and Discussion: We have provided additional structural discussion regarding the results and analysis related to APRI and added considerations regarding finding similar conditions in clinical practice. The results table has also undergone adjustments to make it more straightforward. Enclosed is the revised manuscript with tracked changes marked up the specific changes made in response to the reviewer's suggestions. These revisions address the reviewer's concerns and improve the manuscript's quality. We value the reviewer's time and effort and believe our manuscript now meets the high standards of F1000 Research. Thank you for considering our resubmission. We look forward to your response and the potential approval of our work for full publication. Kind Regards, Authors Suggestions from the reviewer: 1. Add “patient with chronic hepatitis B” in study title, because population in this study only include patient with hepatitis B. Author response: Thanks for the suggestion. We have added information about Chronic Hepatitis B patients in the study title. 2. Elaborate further regarding why APRI cut off for diagnosing liver fibrosis (0.311) is lower than many previous studies Author response: Thanks for the suggestion. After reviewing and comparing various articles, we have added arguments related to the APRI cut-off value from our analysis to the discussion section. 3. More explain why APRI was excluded from the model. Author response: Thanks for the suggestion. The strong correlation with FIB-4 and weaker overall performance than FIB-4, obtained from the prefix analysis, would make APRI insignificant if included in the statistical analysis. 4. Regarding study title “stepwise diagnosis analysis” I would suggest the author to provide how to apply study result to real life practice Author response: Thanks for the suggestion. We have added the steps to follow when applying our study results in real-life practice. This information is available at the end of the discussion section. 5. Minor revision maybe it would be better if in table 2, in variable APRI, FIB-4, AAR and M2BPGi row, add note which one is lower or higher than cut off level Author response: Thanks for the suggestion. We added information to Table 2 to make it more straightforward." } ] }, { "id": "286137", "date": "27 Jun 2024", "name": "Chyntia Olivia Maurine Jasirwan", "expertise": [ "Reviewer Expertise gastroenterology and hepatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigates the use of Mac-2-binding protein glycosylation isomer (M2BPGi) as a diagnostic marker for liver fibrosis in chronic hepatitis B (CHB) patients. The study aims to determine the optimal cut-off values for M2BPGi and other non-invasive fibrosis markers (APRI, FIB-4, and AAR) and assess the diagnostic performance of these markers, both individually and in combination through a stepwise diagnostic analysis.\nThis manuscript clearly presents objectives, methods, results, and conclusions, includes a comprehensive literature review that situates the study within the context of existing research, and accurately and appropriately cites relevant studies.\nSuggestions :\n\n- Abstract: Briefly mention the statistical methods used in the analysis.\n\n- Introduction: Add a few sentences highlighting the specific advantages of non-invasive methods over liver biopsies.\n\n- Discussion of Limitations: Provide a more detailed discussion of the study's limitations, such as the cross-sectional design, potential biases, and generalizability of the findings.\n\n- Future Directions: Suggest specific areas for future research, such as validating the findings in larger, diverse populations.\n\n- Analytical Code: Provide the exact scripts or commands used in SPSS and STATA for the analyses. This can be included as supplementary files.\n\nQuality Control Measures: Please mention any quality control measures or calibration procedures for the laboratory tests and liver elastography.\n\n- Supplementary Material: Include supplementary material detailing the step-by-step process of data processing and analysis.\n\nConfidence Intervals: Include confidence intervals for key statistics such as AUC values and cut-off points to provide a sense of the estimates' precision.\n\n- Validation: Discuss the potential for external validation of the findings in other populations or settings.\n\n- Subgroup Analysis: Perform and discuss subgroup analyses (e.g., based on age and gender) to provide additional insights.\n\n- Processed Data: Include processed data and the analytical code used for statistical analyses.\n\n- Detailed Data Files: Ensure data files are well-documented with clear labels and descriptions of each variable.\n\n- Reproducibility Checklist: Provide a checklist or protocol outlining all steps from data collection to final analysis.\n\n- Limitations: Expand on the limitations section to provide a balanced perspective.\n\n- Future Research Directions: Include specific suggestions for future research based on the study’s findings.\n\n- Confidence Intervals and Effect Sizes: Include these in the results to provide a clearer sense of the precision and reliability of the estimates.\nConclusion The manuscript is scientifically sound, with a clear presentation, appropriate study design, and robust statistical analysis. Minor enhancements, such as providing detailed analytical code, discussing limitations more explicitly, and suggesting future research directions, will further strengthen the manuscript. Addressing these points will ensure the article meets high standards of scientific rigor and transparency.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11992", "date": "12 Jul 2024", "name": "MUHAMMAD BEGAWAN BESTARI", "role": "Author Response", "response": "The F1000 Research Dear Editor and reviewer (Dr Chyntia Olivia Maurine Jasirwan), Subject: Submission of Revised Manuscript I hope this comment finds you well. I am writing to express my sincere gratitude for the feedback provided by the reviewer on our manuscript, “Revisiting Mac-2-Binding Protein Glycosylation Isomer (M2BPGi) for Diagnosing High-Risk Liver Fibrosis: A Stepwise Diagnostic Analysis.” The insightful comments and suggestions have significantly enhanced the quality and clarity of our work. In response to the reviewer's feedback, we have carefully addressed each comment and made substantial revisions to improve the overall quality of the manuscript. The critical revisions include: Limitations and Future Directions: We have added a discussion of potential limitations and future research directions from our research. Research steps, analysis, and results: We have provided additional files for research steps and analysis. The research results and discussion elaboration are presented more thoroughly. Supplementary Files: We have completed and added supplementary files from our paper to support reproducibility and understanding. Enclosed is the revised manuscript with tracked changes marked up the specific changes made in response to the reviewer's suggestions. These revisions address the reviewer's concerns and improve the manuscript's quality. We value the reviewer's time and effort and believe our manuscript now meets the high standards of F1000 Research. Thank you for considering our resubmission. We look forward to your response and the potential approval of our work for full publication. Kind Regards, Authors Suggestions from the reviewer: 1. Abstract: Briefly mention the statistical methods used in the analysis. Author response: Thanks for the suggestion. We have added the statistical methods used in the abstract section. 2. Introduction: Add a few sentences highlighting the specific advantages of non-invasive methods over liver biopsies. Author response: Thanks for the suggestion. In the introduction section, We added a few sentences highlighting the advantages of non-invasive liver fibrosis assessment. 3. Discussion of Limitations: Provide a more detailed discussion of the study's limitations, such as the cross-sectional design, potential biases, and generalizability of the findings. Author response: Thanks for the suggestion. In the discussion section, we have added a more detailed discussion regarding research limitations. 4. Future Directions: Suggest specific areas for future research, such as validating the findings in larger, diverse populations. Author response: Thanks for the suggestion. We have completed the suggestions for future research in the last paragraph of the discussion section. 5. Analytical Code: Provide the exact scripts or commands used in SPSS and STATA for the analyses. This can be included as supplementary files. Author response: Thanks for the suggestion. We have added a reproducibility checklist for the analytical code information to the extended data. 6. Quality Control Measures: Please mention any quality control measures or calibration procedures for the laboratory tests and liver elastography. Author response: Thanks for the suggestion. In the methods section, We added information about the quality control and calibration for the laboratory tests and liver elastography. 7. Supplementary Material: Include supplementary material detailing the step-by-step process of data processing and analysis. Author response: Thanks for the suggestion. We have added a reproducibility checklist to the extended data, detailing the step-by-step process of data processing and analysis. 8. Confidence Intervals: Include confidence intervals for key statistics such as AUC values and cut-off points to provide a sense of the estimates' precision. Author response: Thanks for the suggestion. We have completed the confidence intervals and AUC data found and needed to interpret the research results as a whole. 9. Validation: Discuss the potential for external validation of the findings in other populations or settings. Author response: Thanks for the suggestion. We have completed suggestions for validating our research results in the last paragraph of the discussion section. 10. Subgroup Analysis: Perform and discuss subgroup analyses (e.g., based on age and gender) to provide additional insights. Author response: Thanks for the suggestion. We have added the results of the subgroup analysis in the discussion section to support and explain the overall research results. 11. Processed Data: Include processed data and the analytical code used for statistical analyses. Author response: Thanks for the suggestion. We have completed the processed data in the Data Availability section and the analytical code can be seen in the reproducibility checklist in the extended data. 12. Detailed Data Files: Ensure data files are well-documented with clear labels and descriptions of each variable. Author response: Thanks for the suggestion. We have completed the underlying data with clear labels and descriptions. 13. Reproducibility Checklist: Provide a checklist or protocol outlining all steps from data collection to final analysis. Author response: Thanks for the suggestion. We have added a reproducibility checklist in the extended data for all steps from data collection to final analysis for the Stepwise Diagnostic Analysis. 14. Limitations: Expand on the limitations section to provide a balanced perspective. Author response: Thanks for the suggestion. In the final part of the discussion, we have dissected the potential limitations of our study. 15. Future Research Directions: Include specific suggestions for future research based on the study’s findings. Author response: Thanks for the suggestion. We have completed suggestions for future research directions in the last paragraph of the discussion section. 16. Confidence Intervals and Effect Sizes: Include these in the results to provide a clearer sense of the precision and reliability of the estimates. Author response: Thanks for the suggestion. We ran a categorical predictor variables analysis to form some models that would also predict categorical variables (HR or LR group). Through those analyses, the effect of the model was assessed through Nagelkerke R2, which we have added in the results section. In addition, we have included all important confidence interval data, including CIs for each AUC, to find the cut-off value in the discussion section." } ] } ]	1	https://f1000research.com/articles/13-280
https://f1000research.com/articles/12-1583/v1	14 Dec 23	{ "type": "Research Article", "title": "Depression, anxiety, and stress among urban and rural adolescents in Shivamogga, Karnataka", "authors": [ "Ajay Mallya", "Raghavendraswamy Koppad", "Praveen Kumar", "Raghavendraswamy Koppad", "Praveen Kumar" ], "abstract": "Background: Currently there are 1.3 billion adolescents worldwide, which makes up 16% of the world population. Over 20% of adolescents around the world are thought to have behavioural or mental health issues. Addressing mental health issues is very important for the promotion of positive health in adolescents.\nMethods: The objective of our study was to estimate the prevalence of depression, anxiety, and stress among adolescents in urban and rural areas of Shivamogga. A cross-sectional study was conducted among 350 adolescents aged 16 to 19 years each from urban and rural areas of Shivamogga.\nResults: Depression, anxiety and stress were found to be 23.1%, 29.4% and 26.6% in urban areas and 19.1%, 24% and 21.1% in rural areas respectively.\nConclusions: About a quarter of the adolescent population suffers from depression anxiety and stress. Adopting and implementing better education and health policies are necessary to enhance adolescent mental health.", "keywords": [ "Mental health", "Adolescence", "Depression", "anxiety", "stress" ], "content": "Introduction\n\nCurrently, there are 1.3 billion adolescents worldwide, which makes up 16% of the world population.1 Adolescents make up 20% of the population in countries in the South-East Asia region (SEAR) of the World Health Organization (WHO), and 21.8% of the Indian population.2,3\n\nAdolescence is a distinct period in human development and crucial for setting the groundwork for long-term health. Today nine out of ten adolescents are living in underdeveloped countries, where they confront particularly difficult obstacles in everyday lives from getting an education to simply surviving.4 Over 20% of adolescents around the world are thought to have behavioural or mental health issues. Lifetime mental health disorders commonly begin by the age of 14, and depression is the leading cause of the global disease burden among adolescents.4\n\nAdolescents frequently suffer from emotional disturbances. Anxiety disorders predominate among adolescents and are more frequently seen in older adolescents compared to younger ones. Anxiety disorders are thought to affect 3.6% of 10–14-year-olds and 4.6% of 15–19-year-olds.5 Depression and anxiety share some common features and together can result in poor performance in school and poor school attendance.5 This, in addition to the marked social impairment experienced by depressed adolescents, will result in poor workplace relationships which in turn leads to poor connections and can affect one’s ability to make academic and workplace progress. Depression in adolescents also decreases the chance of them pursuing higher education and can also lead to lower employment rates. Depression in adolescents is also associated with earlier pregnancy and early parenthood.6\n\nAdolescents with depression run a very high risk of suicide.7 Smoking, substance abuse, and obesity are all also more prevalent among adolescents who are depressed.8,9 Depressive disorders are among the top three causes of adolescent disability adjusted life years (DALYs) lost globally and anxiety happens to be among the top five causes of loss of DALYs among adolescent girls.10 About 7% of adolescents between the ages of 13 and 17 suffer from diagnosed mental health disorders in India according to the National Mental Health Survey 2015-16.11\n\nStress in adolescents also affects their academic performance.12 Levels of stress experienced are shown to increase from preadolescence to adolescence.13 Studies have shown that girls tend to experience higher levels of stress compared to boys during adolescence.14 There is evidence that shows a positive correlation between stress and depression and anxiety.15\n\nAddressing mental health issues is very important for the promotion of positive health in adolescents. It also facilitates a whole generation of adults with sound mental health and leads to a healthy society. And as we couldn’t identify any studies done on mental health issues in this region of Karnataka, there is a need to carry out research and gather more data and facilitate the promotion of positive mental health among adolescents in this part of Karnataka.\n\n\nMethods\n\nEthical approval was granted by the Institutional Ethics Committee of Shivamogga Institute of medical Sciences, Shivamogga (ref no. SIMS/IEC/414) on 09/11/2018. Informed consent was taken from study participants and the parents/guardians in case of minors (16-17 years) via Google Forms. Assent was also taken from study participants of age 16-17 years.\n\nA cross-sectional study was conducted in urban and rural areas of Shivamogga taluk to estimate the prevalence of depression, anxiety, and stress among adolescents. This study was conducted from August 2020 to September 2020. The study population is composed of all adolescent males and females aged 16-19 years in rural and urban field areas of Shivamogga taluk. According to an Indian study, the prevalence (p) of anxiety among adolescents was found to be 24.4%.16 With desired absolute precision of 5% the minimum sample size required was calculated using the formula n=4pq/D2. With this, we got a sample size of 295 adolescents. Considering 10% as non-respondents, the final sample size was 325 which was rounded off to 350. So, 350 samples were collected in urban and rural areas each.\n\nThe Block education officer was approached and a list of all pre-university colleges in Shivamogga taluk was collected. Shivamogga taluk has 36 pre-university colleges in the urban area and 10 pre-university colleges in the rural area. Then 50% of the pre-university colleges were randomly selected using the random number table both in urban and rural areas. So, 18 colleges from the urban and 5 colleges from the rural area were selected. From each selected colleges, a class was randomly selected. For each selected class the lottery method was used to select 20 students from each class in the urban area and 70 students from the rural area until the desired sample size was reached. All adolescents in the age group of 16 to 19 years were included in the study. Adolescents who did not fill out the questionnaire completely were excluded from the study.\n\nAfter taking consent, a questionnaire that included basic demographic details (age and sex) with the DASS 21 scale (Depression Anxiety Stress Scale)17 was used to collect data. A Google Form of the questionnaire was created, one each for urban and rural areas and the link to fill out the form was shared with students willing to participate in the study via ‘WhatsApp’ mobile application. Adolescents aged 16 completed years and above until the age of 19 were included in the study.\n\nData was entered into a Microsoft Excel spreadsheet. SPSS (Version 21) was used to perform statistical analysis. Data analysis was done using appropriate statistical tools. Results were expressed in terms of frequency, percentages, Chi-square values, and P-values. All statistical tests were performed with a confidence level of 95% and a power of 80%. In the significance tests, p-values less than 0.05 were considered significant.\n\n\nResults\n\nAs shown in Table 1: a total of 700 adolescents participated in the study. Out of which 350 (50%) adolescents were from urban areas and another 350 (50%) adolescents were from rural areas. Males and females in urban areas were 153 (44%) and 197 (56%) respectively. In rural areas, males and females were 191 (55%) and 159 (45%) respectively.\n\nAll our study participants were aged between 16 to 19 years. the mean age of the participants in the urban area was 17.10±0.84 years. In the urban area, the majority of the participants (142) were aged 17 years (40.6%). Participants aged 16 years were 94 (27%), 18 years were 100 (29%) and 19 years were 14 (4%). Similarly in rural areas, the mean age of the participants was 17.10 ±0.76 years. The majority (159) of the participants were aged 17 years (45%). Participants aged 16 years were 60 (17%), 18 years were 121 (35%) and 19 years were 10 (3%).\n\nAmong the adolescents in urban areas, 23.1% were depressed, 29.4% had anxiety and 26.6% had stress. Similarly, among the adolescents in rural areas, 19.1% were depressed, 24% had anxiety and 21.1% were stressed.\n\nAmong the depressed adolescents, in urban areas the majority (46; 13.1%) had moderate depression, whereas in rural areas the majority (32; 9.1%) had mild depression. Among the adolescents who had anxiety, both in urban and rural areas the majority (71 (20.3%) and 58 (16.6%) respectively) had moderate anxiety. Among the adolescents who were stressed, both in urban and rural areas, the majority (63 (18%) and 54 (15.4%) respectively) had mild stress.\n\nAmong the study participants from urban areas of Shivamogga, 33 (21.6%) adolescent males and 48 (24.4%) adolescent females were depressed, 33 (21.6%) adolescent males and 70 (35.5%) adolescent females were anxious, and 55 (35.9%) adolescent males and 38 (19.3) adolescent females were stressed (see Table 2). A significantly greater number of females were anxious and stressed than males.\n\nAmong the study participants from rural areas of Shivamogga, 26 (13.6%) adolescent males and 41 (25.8%) adolescent females were depressed, 40 (20.9%) adolescent males and 44 (27.7%) adolescent females were anxious, and 47 (24.6%) adolescent males and 27 (17%) adolescent females adolescents were stressed. A significantly greater number of females were stressed than males; see Table 3.\n\n\nDiscussion\n\nOur study attempted to find out the prevalence of depression, anxiety, and stress among adolescents in urban and rural areas. The current study found that the prevalence of depression was 23.13% and 19.14% in urban and rural areas respectively. On average, depression among adolescents in Shivamogga is 21.14%. Similar results were found by Kumar K S et al. and Sahoo S, where depression among adolescents was 19.5%. and 18.5% respectively.16,18 Depression was seen to be higher in females in both urban and rural areas. The difference was statistically significant in rural areas of Shivamogga. Similar observations were made in other studies across India where depression was seen to be higher among female respondents.16,19,20 Genetic predisposition and hormonal influences could be the reason why depression was higher in females than males. This could also be due to higher reporting of symptoms by females, whereas fewer males may report symptoms due to societal pressure for males to be more emotionally strong.\n\nDepression was more frequently seen in urban adolescents than rural adolescents in the current study (23.1% and 19.4% respectively). Similar observations were made by Bahl R, and Kumari R in their study, where depression among urban adolescents was about 33.3% and depression among rural adolescents was 20.7%.21 This could be due to higher exposure of adolescents to risk factors like academic competition and failure, anxiety, and substance abuse, etc.\n\nAnxiety among adolescents in the current study was found to be 29.2% and 24% in urban and rural areas of Shivamogga respectively. On average, anxiety was found to be 26.6%. This was similar to the study conducted by Kumar K S et al. and Madasu S et al., who found anxiety among adolescents to be 24.4% and 22.7% respectively.16,22 These studies also found that the prevalence of anxiety was higher among females when compared to males. Similar findings were seen in our study in both urban and rural areas. The prevalence of anxiety was significantly higher among females than males in urban areas. Higher anxiety among females could be due to higher exposure to risk factors like academic competition and societal pressure on women to work and prove themselves as equals to males in the modern world.\n\nAnxiety was more frequently seen in urban adolescents than rural adolescents in our study (29.4% and 24% respectively). Similar results were observed by Vs P et al.23 This could be due to more academic competitiveness in an urban area than in a rural area. While the parental expectation and societal pressure to perform better academically and excel in the future and have a well-paying job is higher for males than females, in the modern world the same pressure is higher in females as well in urban areas. This could be the reason why anxiety was more prevalent among urban females than males. As this study was conducted during the pandemic, the increased awareness of COVID in urban areas with better education could also be the reason why anxiety was higher among urban adolescents.\n\nStress among the adolescents in our study was found to be 26.6% and 21.1% in urban and rural areas respectively. On average 23.85% of the adolescents were stressed in Shivamogga. Sahoo and Saddicha, in their study, also found that the prevalence of stress among adolescents was 20%.18 Prevalence of stress was found to be higher in urban areas compared to rural areas. Similar observations were made by Dey BK24 in Bangladesh. No studies from the Indian setting could be found that compared the prevalence of stress in urban and rural areas.\n\nStress was more frequently seen in males than females in both urban and rural areas of Shivamogga. This was contrary to observations made in other studies, where they found that stress was more prevalent among female participants.16,24 This difference could be due to underreporting of symptoms by female participants. The ongoing pandemic at the time of the study could have contributed to these findings.\n\nThe strength of the study lies in its robust methodology, extensive sample size and comprehensive data analysis. This study employed a well-designed survey or assessment tool to gather data, ensuring the accuracy and reliability of the results. The measurement of depression, anxiety, and stress was conducted using a standardized scale rather than through assessment by a trained psychiatrist.\n\n\nConclusions\n\nAbout a quarter of the adolescent population suffers from depression anxiety and stress. It’s crucial to detect these symptoms among adolescents and take proper action to avoid compromising their education and development. Those detected to have these symptoms must be referred to be seen by a psychiatrist for clinical evaluation. Further studies are necessary to learn about the causative factors of these mental health disorders. Adopting and implementing better education and health policies are necessary to enhance adolescent mental health.", "appendix": "Data availability\n\nfigshare: DASS.xlsx. https://doi.org/10.6084/m9.figshare.23652477.v2 25\n\nThis project contains the underlying data file.\n\nfigshare: Questionnaire - Depression, anxiety, and stress among adolescents in Shivamogga, Karnataka. https://doi.org/10.6084/m9.figshare.23996670.v2. 26\n\nThis project contains the blank questionnaire.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAdolescents Statistics: UNICEF DATA.[cited 2023 Apr 1]. Reference Source\n\nAdolescent health: [cited 2023 Apr 1]. Reference Source\n\nBehal S, Debarati M, Oommen K: Investing in Adolescent Health: Harnessing India’s Demographic Dividend. ORF. [cited 2023 Apr 1]. Reference Source\n\nUNICEF, editor. Adolescence: an age of opportunity. New York, NY: UNICEF; 2011; 138 p. (The state of the world’s children).\n\nMental health of adolescents: [cited 2023 Apr 1]. Reference Source\n\nSystematic Review and Meta-Analysis: Adolescent Depression and Long-Term Psychosocial Outcomes - ClinicalKey.[cited 2023 Apr 1]. Reference Source\n\nWindfuhr K, While D, Hunt I, et al.: Suicide in juveniles and adolescents in the United Kingdom. J. Child Psychol. Psychiatry. 2008; 49(11): 1155–1165. PubMed Abstract \| Publisher Full Text\n\nHasler G, Pine DS, Kleinbaum DG, et al.: Depressive symptoms during childhood and adult obesity: the Zurich Cohort Study. Mol. Psychiatry. 2005 Sep 1; 10(9): 842–850. PubMed Abstract \| Publisher Full Text\n\nKeenan-Miller D, Hammen CL, Brennan PA: Health Outcomes Related to Early Adolescent Depression. J. Adolesc. Health. 2007 Sep; 41(3): 256–262. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Global accelerated action for the health of adolescents (AA-HA!): guidance to support country implementation. Geneva: World Health Organization; 2017 [cited 2023 Apr 1]; 176. Reference Source\n\nNIMHANS: National Mental Health Survey of India, 2015-16: Mental Health Systems. National Institute of Mental Health and Neuro Sciences; 145. Reference Source\n\nMoksnes UK, Moljord IEO, Espnes GA, et al.: The association between stress and emotional states in adolescents: The role of gender and self-esteem. Personal. Individ. Differ. 2010 Oct 1; 49(5): 430–435. Publisher Full Text\n\nRudolph KD: Gender differences in emotional responses to interpersonal stress during adolescence. J. Adolesc. Health. 2002 Apr; 30(4): 3–13. PubMed Abstract \| Publisher Full Text\n\nArsenio WF, Loria S: Coping with Negative Emotions: Connections with Adolescents’ Academic Performance and Stress. J. Genet. Psychol. 2014 Jan 1; 175(1): 76–90. Publisher Full Text\n\nSex Differences in Adolescent Depression: Stress Exposure and Reactivity Models - Hankin - 2007 - Child Development - Wiley Online Library.[cited 2023 Apr 17]. Publisher Full Text\n\nKumar KS, Akoijam BS: Depression, Anxiety and Stress Among Higher Secondary School Students of Imphal, Manipur. Indian J. Community Med. 2017; 42(2): 94–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDepression Anxiety Stress Scales (DASS): [cited 2023 Sep 8]. Reference Source\n\nSahoo S, Khess CRJ: Prevalence of Depression, Anxiety, and Stress Among Young Male Adults in India: A Dimensional and Categorical Diagnoses-Based Study. J. Nerv. Ment. Dis. 2010 Dec; 198(12): 901–904. Publisher Full Text\n\nSinghal M, Manjula M, Vijay Sagar KJ: Subclinical depression in Urban Indian adolescents: Prevalence, felt needs, and correlates. Indian J. Psychiatry. 2016; 58(4): 394–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMishra SK, Srivastava M, Tiwary NK, et al.: Prevalence of depression and anxiety among children in rural and suburban areas of Eastern Uttar Pradesh: A cross-sectional study. J. Family Med. Prim. Care. 2018; 7(1): 21–26. Publisher Full Text\n\nBahl R, Kumari R: Mental Health Profile of Urban and Rural Adolescents in Jammu District of J&K.2016; 18: 3.\n\nMadasu S, Malhotra S, Kant S, et al.: Anxiety Disorders among Adolescents in a Rural Area of Northern India using Screen for Child Anxiety-Related Emotional Disorders Tool: A Community-based Study. Indian J. Community Med. 2019; 44(4): 317–321. PubMed Abstract \| Publisher Full Text\n\nPrabha VS, Rao VB, Kanakabushanam GVVS: A comparative study of anxiety and depression among adolescents from rural and urban areas. J. Med. Sci. Res. 2017 Jan 3; 5(1): 29–32. Publisher Full Text\n\nDey BK, Rahman A, Bairagi A, et al.: Stress and Anger of Rural and Urban Adolescents. Psychology. 2014 Mar 18 [cited 2023 Apr 23]; 2014. Reference Source\n\nMallya A: Responses - Depression, anxiety, and stress among adolescents in Shivamogga, Karnataka. [Dataset]. figshare. 2023. Publisher Full Text\n\nMallya A: Questionnaire - Depression, anxiety, and stress among adolescents in Shivamogga, Karnataka. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "257925", "date": "23 Mar 2024", "name": "Indranil Saha", "expertise": [ "Reviewer Expertise Epidemiology", "Non-communicable disease", "Mental health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript needs following clarifications:\n“p-values less than 0.05 were considered significant” – please correct as p value equal to or less than 0.05 was considered as statistically significant As per rule the adolescents with completed 19 years should have been included as study participants Colleges situated in urban and rural area: whether urban college truly represent urban adolescents? There may be so may students from rural area studying in urban college and the vice versa may be true.\n\nHow socioeconomic status was measured in both the colleges? Please edit the sample size formula – “n=4pq/D2” instead of 4, please mention as Z∞2. In which language this DASS 21 scale was used? Was it validated and reliable in that settings?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "11396", "date": "21 Jun 2024", "name": "Ajay Mallya", "role": "Author Response", "response": "Comment: “p-values less than 0.05 were considered significant” – please correct as p value equal to or less than 0.05 was considered as statistically significant Author’s Response: We agree with your comment. The suggested correction has been made. Action Taken in the manuscript: The suggested correction has been made. Comment: As per rule the adolescents with completed 19 years should have been included as study participants Author’s Response: We agree with your comment. We would like to clarify that adolescents who have completed 19 years were included in the study. Action Taken in the manuscript: we have mentioned that adolescents aged 19 years have included in the study in the methodology section. Comment: Colleges situated in urban and rural area: whether urban college truly represent urban adolescents? There may be so may students from rural area studying in urban college and the vice versa may be true. Author’s Response: In the data collection process, we ensured that the collected data is properly segregated into urban and rural categories based on the list of colleges obtained from the block education officer. However, we acknowledge that some participants may commute and study in a college that does not accurately represent the demography (urban and rural). This can be seen as a limitation of the study. Action Taken in the manuscript: Necessary changes have been done in the strengths and limitation section. Comment: How socioeconomic status was measured in both the colleges? Author’s Response: We have not measured the socioeconomic status of the participants in the study. Action Taken in the manuscript: As socioeconomic status was not included in the original study instrument we have not included it in the study. Comment: Please edit the sample size formula – “n=4pq/D2” instead of 4, please mention as Z∞2 Author’s Response: We agree with your observation. Action Taken in the manuscript: Necessary changes have been done in the manuscript. Comment: In which language this DASS 21 scale was used? Was it validated and reliable in that settings? Author’s Response: DASS21 Scale was used in English in our study without any translation or alteration. As the students studying in PU college have English as their first language we used the scale without any translations. DASS21 scale in English is already validated among adolescents. Action Taken in the manuscript: We have duly incorporated this information into the methodology section of the manuscript for clarity." } ] } ]	1	https://f1000research.com/articles/12-1583
https://f1000research.com/articles/13-459/v1	08 May 24	{ "type": "Systematic Review", "title": "Statistical data transformation in agrarian sciences for variance analysis: a systematic review", "authors": [ "Jhennifer Nascimento", "Jonas Silva", "Rodrigo Cupertino Bernardes", "Guilherme Costa", "Paulo Emiliano", "Jonas Silva", "Rodrigo Cupertino Bernardes", "Guilherme Costa", "Paulo Emiliano" ], "abstract": "In statistical analyses, a common practice for enhancing the validity of variance analysis is the application of data transformation to convert measurements into a different mathematical scale. This technique was first employed in 1898 by Edgeworth and remains relevant in current scientific publications despite the proliferation of more modern and advanced techniques that obviate the need for certain assumptions. Data transformations, when appropriately used, can make the model error terms approximate a normal distribution. It is also possible to use the technique to correct the heterogeneity of variances or to render an additive model, ensuring the validity of the analysis of variances. Given that this technique can be hastily applied, potentially leading to erroneous or invalid results, we conducted a systematic literature review of studies in the field of agrarian sciences that utilized data transformations for the validation of analysis of variances. The aim was to check the transformations employed by the scientific community, the motivation behind their use, and to identify possible errors and inconsistencies in applying the technique in publications. In this study, we identified shortcomings and misconceptions associated with using this method, and we observed incomplete and inadequate utilization of the technique in 94.28% of the analysed sample, resulting in misguided and erroneous conclusions in scientific research outcomes.", "keywords": [ "Data manipulation", "Systematic literature review", "Agricultural sciences", "ANOVA", "Descriptive analysis" ], "content": "1. Introduction\n\nThe analysis of variance (ANOVA) is a method used to test the hypothesis of statistical equality among a set of population means, which is a superior option to the t-test due to the increased probability of committing a type I error when using it for comparing multiple means, as stated by Mohr.1 To validate the ANOVA analysis, assumptions such as additivity of treatment and environmental effects, independence, homoscedasticity of variances, and normality of experimental errors need to be satisfied, as pointed out by Cochran.2 Data transformations can be applied to validate the analysis when these assumptions are unmet.\n\nInitially proposed by Edgeworth3 and referred to as the Translation Method, data transformations emerged from the need to relate observed distributions to the normal distribution. According to Bartlett,4 the usual purpose of transformation is to change the scale of measurements to make the ANOVA analysis valid. However, a transformation is successful only if it satisfies the requirements of variance homogeneity, additivity structure, and error normality through the same transformation, as stated by Atikinson.5\n\nAfter any transformation, verifying whether it has improved the data distribution is essential, as highlighted by Quinn and Keough.6 Besides that, data transformation provides an accessible solution to avoid non-normal error distributions, as stated by Pierre et al.,7 allowing for easy analyses by applying linear models. However, researchers must exercise caution when using transformations, as they can have contrary effects, Rupert.8\n\nDespite frequently using data transformations in scientific literature, they receive criticism from theoretical and practical perspectives, Oliveira et al.9 One of the main criticisms of data transformations is the potential to alter the interpretation of results, Box and Cox.10 According to these authors, data transformation can change the focus of the study, leading to different conclusions than those obtained from the original data. Additionally, some transformations may not be readily interpretable, hindering the communication of results due to the transformed scale. Another criticism is that transformations can affect the robustness of ANOVA, resulting in false positives or negatives, Hocking.11 Therefore, carefully considering the implications of any data transformation before applying it in a variance analysis is crucial.\n\nDue to the large volume of studies in agrarian sciences that conduct variance analyses and can make use of various data transformations to validate them, a systematic literature review is a valuable approach to identifying subject trends and determining whether there is proper utilization of transformation techniques to ensure reliable and valid results of variance analysis. According to Khan,12 a systematic review involves systematically collecting and examining published works to extract relevant statistics and information from the selected studies. On the other hand, meta-analysis is the quantitative aspect of synthesizing the analysed works, allowing for a numerical summary of the observed results.\n\nIn this context, we conducted a systematic literature review of published works in agrarian sciences that employ data transformations to validate variance analyses. We performed a detailed analysis of various publications that utilized this technique to identify the most common transformations adopted by the scientific community and the motivations behind their use. This study also identified shortcomings and misconceptions associated with applying this method. The results of the analysed sample revealed an inadequate application of the technique by the scientific community, highlighting the need for enhanced methodological rigor in such analyses by researchers.\n\n\n2. Methods\n\nThe systematic literature review process adopted in this study followed the guidelines of PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses), developed by Moher et al.13 These guidelines describe the stages of identification, screening, eligibility, and inclusion, ensuring the results’ validity and reliability.\n\nIn the identification stage, studies of interest are located in the databases, followed by selecting and classifying works based on titles and abstracts in the screening stage. In the eligibility stage, we evaluate the full text of the selected articles from the previous stage. In the inclusion stage, relevant information is systematically extracted based on predefined topics.\n\nHowever, our methodology adapted to these guidelines in the screening phase. Instead of analysing only the articles’ abstracts, we downloaded the selected articles in the identification stage. We searched for specific terms in the full text of these works to select the articles that would proceed to the eligibility and inclusion stages. This strategy was employed because crucial information about the transformations is sometimes present in the abstracts, title, and methodology. This way, the information of interest would be noticed.\n\n2.1.1 Identification stage\n\nTo identify articles in the field of agricultural sciences that have conducted variance analyses, we conducted comprehensive searches on academic platforms such as Scopus and Web of Science, using keywords such as “variance analysis,” “analysis of variance,” and “ANOVA.” We narrowed our search to articles published exclusively in English, the global language of academia and research. This approach ensures a uniform and accessible foundation for the chosen studies.\n\nDue to the absence of specific filters for agricultural sciences in the platforms used, we employed different refinement approaches for each platform. In Scopus, under the “subject area” tab, we filtered by “Agricultural and Biological Sciences,” excluding journals unrelated to agricultural sciences. In Web of Science, under the “Web of Science Categories” tab, we filtered by “Agronomy,” “Agriculture Dairy Animal Science,” “Agriculture Multidisciplinary,” “Agricultural Engineering,” and “Agricultural Economics Policy.” The last search for each source was conducted on May 14th.\n\n2.1.2 Screening stage\n\nDue to the high quantity of files to be downloaded and analysed, we only worked with open-access files. We exported these files in the RIS format to Rayyan, a web application developed by the Qatar Computing Research Institute for systematic reviews. We organized the files according to the publication year and manually excluded duplicates to prevent unnecessary file downloads.\n\nWe utilized the “pyautogui,” “openly,” and “pandas” libraries to develop a Python automation that downloads the files in pdf format directly from Scopus and Web of Science platforms. The ones not downloaded through automation were obtained manually.\n\nSubsequently, we employed the “pypdf4” library to develop a Python search code to search for the terms “data were transformed,” “transformation of the data,” and “data transformation” within the full texts of the works, aiming to reduce the number of articles required for reading and analysis.\n\nPython is freely available software accessible at https://www.python.org/, and the Python code can be found at https://github.com/ghscosta/data-transform for anyone interested in replicating the procedures outlined in this article.\n\n2.1.3 Eligibility and inclusion stages\n\nDuring the eligibility stage, two reviewers examined the complete texts of the articles resulting from the screening process and extracted relevant information in the inclusion phase. We carried out these two stages simultaneously. From the articles that underwent the initial filtering process, we collected the following information: reference number for article identification, title, journal, year of publication, authors, utilization of analysis of variance in the methodology, employed design, verification of ANOVA assumptions, type of applied transformation, rationale for the adoption of the transformation, re-evaluation of assumptions after data transformation, scale of interpretation for results, study element in the experiment, and impact factor of each journal. The description of each variable can be accessed in Table 1, available at https://rpubs.com/JhenniferNascimento/table and https://zenodo.org/records/10757759. We discarded articles that did not provide this information and considered only those containing the pertinent details for the descriptive analysis.\n\nTo present the collected data and potential relationships among variables, we employed bar graphs, pie charts, and Sankey diagrams for representing and analysing the information obtained from the sampled articles. We manually tabulated the data, and a concise summary is provided in our extended data, accessible in Table 2 at https://rpubs.com/JhenniferNascimento/table and https://zenodo.org/records/10757759.\n\nPie and bar charts were used to illustrate the eligibility criteria for determining the inclusion of studies in each synthesis, along with the percentages of discarded and included studies. Sankey diagrams were employed to depict key variables and elucidate potential relationships among them.\n\n\n3. Results\n\n3.1.1 Identification stage\n\nThe search for the terms “variance analysis,” “analysis of variance,” and “ANOVA,” which we conducted on the Web of Science and Scopus platforms, resulted in retrieving $8,460$ files on the former and 8,695 on the latter. Out of this total, 3,615 files from Web of Science and 3,529 from Scopus were available in open access. Consequently, during the identification stage, we obtained a combined set of 7144 articles published between 1961 and 2023 from both platforms.\n\n3.1.2 Screening stage\n\nAfter the removal of duplicates, 6,077 articles remained for downloading. We downloaded 5,032 articles through automation, leaving 1,045 for manual download. Despite applying the open access filter on both platforms, we could not access 52 of the articles earmarked for manual downloading, resulting in a total of 6,025 articles successfully downloaded.\n\nBy searching for the terms “data were transformed,” “transformation of the data” and “data transformation” within the full text of the 6,025 articles, we narrowed down the number of articles requiring scrutiny from 6,025 to 565 for analysis in the upcoming stages.\n\n3.1.3 Eligibility and inclusion stages\n\nWe read and collected relevant information from 565 articles, resulting from the previous stages. Among these, 506 articles were discarded, with details about the discards presented in the descriptive analysis. We effectively collected information from only 59 articles relevant to our search.\n\nSome of the tabulated articles conducted more than one experiment in their work or used more than one transformation, thus leading to more than one variance analysis. Each distinct experiment and transformation were treated as separate observations to ensure correct counting. As a result, the sample of 59 articles generated 70 tabulated observations. The study characteristics are presented in Table 2, available at https://rpubs.com/JhenniferNascimento/table.\n\nFigure 1 presents the quantities of articles discarded and included for tabulation, where the stacked bars visually show the total of 506 and 59 articles discarded and included in the final sample, respectively. On the other hand, the first pie chart presents the reasons used for the discard, and the second one shows the transformations used for the validation of the analysis of variances.\n\nIn the first circular sector, it is evident that we discarded approximately 78% of the articles from fields like medicine, engineering, psychology, and physical education because they were outside the field of agrarian sciences. We discarded approximately 15% of the articles for not containing variance analysis, 4.55% for not using data transformation, and just over 1% were books and, therefore, incompatible with the inclusion criteria.\n\nIn the second circular sector of Figure 1, which relates to articles with relevant information for our study published between 1994 and 2022, we observed five types of transformations. These include logarithmic, square root, Box-Cox, power, and arcsine transformations, used to validate variance analysis in experiments involving crops such as corn, rice, passion fruit, and others, as illustrated in Figure 2. We grouped categories with only one occurrence as “other” for the “study element” variable.\n\nIn the “Other” category, we have study elements that had only one occurrence in the sample.\n\nIn addition to examining the relationship between the study elements of the experiment and the transformations used, we investigated using Figure 3 to explore the interactions and connections among the transformations. We also verified assumptions, assessed the motivation for employing these transformations, considered the interpretation scale, and reevaluated the assumptions of analysis of variance after applying the transformation.\n\nAs a result, Figure 3 highlights that transformations were applied within the final sample with (55.71%) and without (44.28%) the verification of analysis of variance assumptions. Among the sampled studies that checked the assumptions, 66.66% verified at least one of the three assumptions (normality, homogeneity, and independence of residuals) before applying the transformation, while 33.33% mentioned the verification of assumptions but did not specify which the checked assumption.\n\nAmong the observed transformations in the sample (as illustrated in Figure 3), logarithmic (28.57%), arcsine (21.42%), and square root (20%) transformations were the most used, regardless of whether checked assumptions. At the same time, 18.57% did not specify the transformation used.\n\nFigure 3 also reveals the presence of articles that did not verify assumptions, did not specify the transformation used, did not record the rationale behind the choice of transformation, and did not report whether the meted assumptions of analysis of variance after the application of the transformation. Only 5.71% of the studies conducted verification of normality, homogeneity, and independence of residuals assumptions and adopted transformations with plausible justifications and motivations, illustrating the technique’s incomplete and potentially incorrect application in 94.28% of the considered studies.\n\nFinally, Figure 3 also provides relevant information regarding the interpretation approach adopted in the studies. Most articles interpreted their results on the transformed scale, considering the data after applying transformations. However, some studies mentioned inverse transformation to revert the data to the original scale before interpretation, which is an incorrect practice. As for the reevaluation of assumptions after the implementation of transformations, most studies did not mention this step.\n\n\n4. Discussion\n\nExecuting the steps proposed by PRISMA (identification, screening, eligibility, and inclusion), with the implemented adaptations, presents an innovative approach to searching and selecting articles for this study. The direct search for the terms of interest in the full text represented a bold strategy, one that could potentially serve as a valuable technique for future systematic reviews, particularly when researchers seek information that may not be explicitly stated in the title or abstract, as was the case in our study.\n\nThe screening phase, implemented with automated article downloading and the search for terms in the complete text, programmed in the Python language, efficiently directed us to the most relevant articles with minimal manual effort required. Although some articles required manual downloading, we significantly reduced the overall effort.\n\nDuring the eligibility and inclusion phases, we identified and selected articles that reported essential information about the implementation of data transformations to validate the analysis of variance. We notice that many of these studies did not unsurprisingly document several relevant pieces of information.\n\nAs depicted in Figure 1, a large portion of the articles was discarded, which is common in systematic literature reviews, as pointed out by Gerstner.14 According to the authors, essential information is often omitted in scientific papers, leading to the exclusion of these studies during the systematic review or meta-analysis stage. In our study, only 10% of the sample provided the information of interest, with the majority of exclusions occurring because they were unrelated to the agrarian sciences field. This result was surprising since we expected to obtain a more representative sample of the field due to the filtering criteria employed on the platforms.\n\nWhen examining the transformations employed in the studies concerning the analysed elements in the experiments (as exemplified in Figure 2), no specific pattern emerged that linked a particular type of element to a specific transformation. By expanding the sample, it is conceivable that we may eventually discern a relevant pattern between the types of elements used in experiments and their interaction with specific transformations.\n\nRegarding the interactions and connections of the variables presented in Figure 3, concerns arise about the validity of the analyses conducted in studies where 55% of the sample did not verify essential assumptions (normality, homogeneity, and independence of residuals) and opted for data transformations. The practice of transforming data without first determining whether it is necessary for the analysis compromises the interpretation and reliability of the results obtained.\n\nOn the other hand, the finding that 26 out of 39 articles that effectively assessed at least one of the three assumptions before applying transformations (equivalent to 66.66%), representing only 37.14% of the total sample (of 71 articles), raises questions about the awareness and statistical rigor of authors in handling such data analysis. In contrast, the 33.33% of articles mentioning assumption verification without specifying which ones indicate a need for more transparency in statistical analysis practices.\n\nThe distribution of data transformations in the sample draws attention to the prevalence of logarithmic, arcsine, and square root transformations, regardless of assumption verification. However, the omission of transformation cases raises concerns about research transparency and reproducibility, underscoring the need for comprehensive documentation of methods to ensure accurate evaluation and replicability.\n\nThe finding that only a tiny portion of the studies adopted transformations with plausible justifications after verifying the assumptions of normality, homogeneity, and independence of residuals, coupled with the presence of studies that did not verify assumptions, did not mention transformations used and did not document justifications for their choices, raises questions about the statistical rigor of authors in conducting such analyses. It highlights the need for greater statistical rigor within the academic community and more explicit guidelines for conducting robust and transparent statistical analyses, which ensure reliable and meaningful results for advancing research.\n\nFurthermore, the highlighted findings also raise essential considerations about academic journals’ peer review and publication process. The journal with the highest impact factor in the sample includes one of the articles that did not verify statistical assumptions, omitted details about applied transformations, and failed to document the rationale behind their choices. Accepting works with such characteristics impacts the quality of scientific literature and compromises the integrity of presented results and the correct interpretation of conclusions.\n\nTherefore, readers of these works often need more evidence to determine the reliability of the executed analysis and the result’s veracity, having to rely on the content presented without the opportunity to develop their conclusions based on the analyses. It shows the importance of a complete approach in describing the procedures used in scientific works that are published and that journals need to be more careful, especially when it comes to implementing this methodology that is so old and used.\n\nRegarding aspects related to result interpretation and the methodological approach adopted in the studies in our sample, the observation that most articles chose to interpret their results on the transformed scale points to an ordinary and correct practice in the literature. However, returning the data to its original scale before interpretation raises a discussion about the validity of this approach, as it can distort the correct interpretation of results and potentially lead to erroneous conclusions. For the articles interpreted in both scales, those made an unnecessary effort. Interpreting must always occur on the transformed scale.\n\nFinally, another relevant point is the lack of mention of reevaluating assumptions after applying transformations. It raises questions about researchers’ awareness of the importance of verifying whether the assumptions of analysis of variance are met even after data transformation. Omitting this step can compromise the reliability and validity of conclusions. The documentation and detailed description of methods used in all scientific research is crucial, especially in the field of agricultural sciences, which was the focus of our study.\n\n\n5. Conclusions\n\nWe introduced a novel approach to screening systematic literature reviews, which combines downloading and searching for the terms of interest directly within the full-text articles. This technique proves valuable, mainly when research of interest is not readily available in titles or abstracts.\n\nThe descriptive analysis of transformations used in the articles revealed the prevalence of logarithmic, arcsine, and square root transformations. It became evident that verifying assumptions before applying transformations was only consistently conducted in some cases, emphasizing the need for a more detailed and consistent approach in research planning and reporting procedures.\n\nInterpreting results on the transformed scale was the most common approach, although some studies adopted reverse transformations to return data to the original scale. However, the omission of analysis details underscores the need for greater clarity and consistency in documenting the procedures employed.\n\nGiven our focus on the field of agricultural sciences and the identified inconsistencies in the technique’s application, questions arise regarding the appropriate use of this method in other areas of knowledge.", "appendix": "Data availability\n\nAll underlying data are available as part of the article and no additional source data are required.\n\nZenodo: Extended data for ‘Statistical data transformation in agrarian sciences for variance analysis: a systematic review’, https://doi.org/10.5281/zenodo.10519177. 15\n\nZenodo: PRISMA checklist and flow diagram for ‘Statistical data transformation in agrarian sciences for variance analysis: a systematic review’, https://doi.org/10.5281/zenodo.10758186. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0 International).\n\n\nReferences\n\nMohr DL, Wilson WJ, Freund RJ: Statistical methods. 4th ed.Chennai: Academic Press; 2021.\n\nCochran WG: When the assumptions for the analysis of variance are not satisfied. Biometrics. 1947; 3(1): 22–38. Publisher Full Text\n\nEdgeworth FY: A suggestion for using powerful and informative tests of normality. R. Stat. Soc. 1898; 61(3): 534–544.\n\nBartlett MS: The use of transformations. Biometrics. 1947; 3(1): 39–52. Publisher Full Text\n\nAtikinson AC: Plots, transformations, and regression. 1st ed.USA: Oxford Science Publication; 1985.\n\nQuinn GP, Keough MJ: Experimental design and data analysis for biologists. 1st ed.New York: Cambridge University Press; 2002.\n\nPierre AP, Shikon V, Schneider DC: Count data in biology - Data transformation or model reformation? Ecol. Evol. 2018; 8(6): 3077–3085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRupert D: Statistical analysis, special problems of transformations of data. Soc. Behav. Sci. 2001; 20(1): 15007–15014.\n\nOliveira JPR, Santana DG, Pereira VJ, et al.: Data transformation: an underestimated tool by inappropriate use. Acta Scientiarum. 2018; 40(1): 35300–35311. Publisher Full Text\n\nBox GEP, Cox DR: An analysis of transformations. J. R. Stat. Soc. 1964.11; 26(2): 211–243. Publisher Full Text\n\nHocking RR: The Analysis and Selection of Variables in Linear Regression. Biometrics. 1976; 32(1): 1–49. Publisher Full Text\n\nKhan S: Meta-analysis: methods for health and experimental studies. 1st ed.Springer; 2020.\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items12for systematic reviews and meta-analyses: The PRISMA Statement. PLoS Med. 2009; 6: e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGerstner K, Mateos DM, Gurevitch J, et al.: Will your paper be used in a meta-analysis? Make the reach of your research broader and longer lasting. Methods Ecol. Evol. 2017; 8: 777–784. Publisher Full Text\n\nNascimento JS, Silva JF, Bernardes RC, et al.: Statistical data transformation in agrarian sciences for variance analysis: a systematic review [data and code].2024.\n\nNascimento JS, Silva JF, Bernardes RC, et al.: Statistical data transformation in agrarian sciences for variance analysis: a systematic review [reporting guidelines].2024." }	[ { "id": "275872", "date": "08 Jun 2024", "name": "Pradip Kumar Sahu", "expertise": [ "Reviewer Expertise Basically I am an Agricultural Statistician having major areas of research as Regression analysis", "Econometrics and Time series forecasting." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 based on 59 articles using transformation in ANOVA is very useful, it could have been still better if 1) in addition to open access articles other articles are also considered 2) in addition to percentage analysis use of other relevant statistical measures could have been more meaningful 3) There is some confusion about sample of articles: written 70 in page 5 and 71 in page 8 4) In page para5 of page 6 details of 88.56% articles have been provided what about rest 11.44% is not clear 5) Information on how many articles have used the transformation properly is of great interest 6) I would be interested to know about the results of test of agreement between the work of two reviewers\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [ { "c_id": "11988", "date": "12 Jul 2024", "name": "Jhennifer dos Santos Nascimento", "role": "Author Response", "response": "Reviewer 1: Pradip Kumar Sahu, Department of Agricultural Statistics, Bidhan Chandra Krishi Viswa Vidyalaya, Nadia, West Bengal, India. Author responses to Reviewer comments are as follows: Reviewer Comment: 1) In addition to open access articles other articles are also considered Author Response: We appreciate the comment. We acknowledge that including closed-access articles would have enriched our study. However, limited financial resources restricted our access to paid articles, hampering our research. We used the articles available on the CAPES journal portal, which the Federal University of Viçosa pays for so that its students have access to various closed-access articles, and even so, there were articles that could not be downloaded. Specifically, we could not access 52 articles from our sample for financial reasons. Please refer to the link (https://www-periodicos-capes-gov-br.ezl.periodicos.capes.gov.br/index.php/sobre/quem-somos.html) for more details about the CAPES journal portal. Reviewer Comment: 2) other relevant statistical measures could have been more significant besides the percentage analysis. Author Response: We agree that the inclusion of other statistical measures would enrich the analysis. We will add the raw values for each category presented in the images to Figure 1, complementing the percentage descriptive analysis. This way, the reader can obtain other statistics they deem relevant. If you have any specific statistical measures you would recommend including, we would be happy to consider them and incorporate them into our analysis. Reviewer Comment: 3) The sample of articles—70 on page 5 and 71 on page 8—needs to be clarified. Author Response: Thanks for pointing out this inconsistency. We corrected a typographical error on page 8. The correct number is 70, as presented on page 5. Reviewer Comment: 4) On page 6, details were provided for 88.56% of the articles, and the remaining 11.44% needs to be clarified. Author Response: The remaining 11.44% refers to articles that used power and box-cox transformations. We will clarify this in the text to avoid confusion. Reviewer Comment: 5) Information on how many articles correctly applied the transformation is fascinating. Information on how many articles correctly applied the transformation would indeed be fascinating. Author Response: Unfortunately, to do so, we would need the raw data from each article to replicate the analyses and determine whether the transformations were correctly applied. Since this data is unavailable, we cannot accurately assess the correctness of the transformations. So, our study focused on analyzing the application of transformations in the articles, not verifying their accuracy. Reviewer Comment: 6) I would like to know the results of the concordance test between the work of two reviewers. Author Response: Please provide more details about what you want to know, specifically about the concordance test between reviewers. This will allow us to address your question more precisely." } ] }, { "id": "287787", "date": "25 Jun 2024", "name": "Pratheesh P Gopinath", "expertise": [ "Reviewer Expertise Design of Experiments", "AI", "R shiny", "R", "Agricultural Statistics", "Social Science", "Climate Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is commendable for its initiative and effort to study data transformation practices in agrarian sciences. It sheds light on the significant issue of improper application of transformations, which can undermine the validity of statistical analyses. The importance of correctly applying transformations cannot be overstated, as it directly impacts the reliability of research findings. The study is particularly interesting as it addresses the critical aspect of when and how data transformations should be applied in statistical analyses. This is of paramount importance in agrarian sciences, where the choice of transformation can significantly influence the results. But the study lacks specificity. While the study does highlight the prevalence of improper transformation applications, it lacks a clear linkage to the motivations behind these practices. Researchers often follow established precedents without conducting normality and homogeneity tests, which is a critical gap that this review should address more explicitly, for example entomology studies mainly employs logarithmic and square root etc. A more detailed field-wise classification of the reviewed studies would enhance the usefulness of this study. Identifying which transformations are used in specific subfields, such as crop science, soil science, and entomology, would provide more actionable insights for researchers. A table showcasing the types of transformations used and their corresponding experimental situations would greatly enrich the paper. This classification would offer a practical guide for researchers on appropriate transformations for their specific research contexts. The presentation of results could be improved. For instance, in pie diagrams instead of decimals commas are used. Clarity of Sankey diagram in conceiving information is also questionable. While descriptive analysis is a useful starting point, it is not sufficient on its own to reveal underlying patterns and issues. The study would benefit from a more in-depth statistical analysis to identify trends and common pitfalls in the application of data transformations. The conclusion part needs to be more specific rather than stressing the novelty of data collection method the concentration can be on the analysis of trend of employing transformations and important conclusion based on that and suggestions.\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? No\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [ { "c_id": "11989", "date": "10 Jul 2024", "name": "Jhennifer dos Santos Nascimento", "role": "Author Response", "response": "Reviewer 2: Pratheesh P Gopinath, Kerala Agricultural University, Kerela, India. We appreciate the positive comments on our study. We agree that the correct application of data transformations is crucial for the reliability of research results in agricultural sciences. As discussed in paragraphs 6, 8, and 10 of section 4.2 of our article, we believe that the inadequate application of statistical techniques, including data transformations, often stems from a lack of statistical rigor in the academic community and the absence of explicit guidelines for conducting robust and transparent statistical analyses. Scientific journals should require authors to provide a complete and detailed description of the statistical procedures used in their work, as accurate documentation and description of methods are fundamental for the replicability and validity of research. Our study could have addressed the motivations behind inadequate data transformation practices more explicitly. As noted in our study, many researchers follow established precedents without performing the necessary normality and homogeneity tests. We intend to investigate this topic more deeply in future studies, including the author's thesis, where we plan to classify in detail the transformations used in specific subfields of agricultural sciences. A detailed classification of the reviewed studies, identifying the transformations used in specific subfields and their experimental situations, would enrich our article. The author continues this work as part of their thesis and plans to publish it in future articles. The researchers aimed to provide an overview of how the academic agricultural sciences community utilizes data transformation techniques. It has yielded valuable insights that we will further explore in subsequent studies. We appreciate the comments on the results presentation. We will correct the commas in the pie charts, replacing them with decimal points. Regarding the Sankey diagram, we chose to use it because it efficiently summarizes the trajectory of data transformations in the analyzed scientific research. The Sankey diagram allows us to present the information compactly and comprehensibly, avoiding the need for multiple bar charts, conditional probability charts, or flowcharts. A bar chart would be necessary for each variable presented, totaling eight bar charts to replace the two presented Sankey charts. If we presented the probability tree of the second Sankey chart, which has six variables with 2, 6, 6, 4, 4, and 4 possibilities, respectively, we would have a tree with 4068 branches (26644*4=4608). We could also have made flowcharts with two variables at a time, totaling 12 flowcharts. Therefore, the Sankey chart summarizes a large amount of data without creating various tables, bar charts, or flowcharts. The reader can calculate any desired conditional probabilities since it presents the raw values. A more in-depth statistical analysis would be beneficial to identify patterns and underlying issues in the application of data transformations. However, this study aimed to provide an overview of data transformation practices in agricultural sciences. The author's thesis investigates the identification of detailed patterns and will address them in future studies. We value the feedback provided on the conclusion. We acknowledge that our current approach emphasizes presenting a method that facilitates systematic review research. However, our conclusion also addressed the results observed in our descriptive analysis, revealing the most used transformations in our sample. We emphasized the need for greater clarity and consistency in the documentation of the procedures used and the inconsistencies identified in applying the technique. For a more specific conclusion, we need to conduct more detailed studies of the subfields of agricultural sciences and the transformations used in each specific area. The author is investigating these issues in their doctoral thesis and plans to address them in future studies. The rationale and objectives of our study were to establish an overview of data transformation practices in agricultural sciences through a systematic review using an adapted screening method. This method facilitates research for researchers who wish to conduct systematic reviews, especially when the information of interest is present in the body of the article but not in the abstract and title. We will improve the last paragraph of the introduction to make this more precise and detailed. The statistical analysis conducted was adequate for this study's objectives, which were to provide an overview of data transformation practices. However, as mentioned earlier, we understand that additional statistical analyses could provide more insights, and this will be addressed in future studies. We based our study's conclusions on the presented results, which aimed to demonstrate an overview of data transformation practices in agricultural sciences. To achieve more detailed and assertive conclusions, we acknowledge the necessity of conducting a more in-depth study currently underway and scheduled for publication in future articles." } ] }, { "id": "275867", "date": "14 Nov 2024", "name": "Manoj Kumar Sharma", "expertise": [ "Reviewer Expertise Applied 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\nComments on the paper “Statistical data transformation in agrarian sciences for variance analysis: a systematic review”.\nThe author (s) have nicely written the proposed paper. It is a unique type of work. This should be widely popularized. The suggestions and comments from my side are given below impactful presentation.\n\n1.The introduction and methods are nicely presented. 2.In the results section the findings are presented only in the form of data-oriented figures. In my point of view, the finding data should also presented in tabular form. 3.In the discussion part some of the results are also presented. So these results should also shifted to the results section. 4.The Conclusion section is nicely presented.\nThe author (s) should be asked to revise the paper as per my suggestions mentioned above. This paper may be accepted after minor revision.\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-459
https://f1000research.com/articles/11-785/v1	13 Jul 22	{ "type": "Systematic Review", "title": "Records of antibodies in breast milk in postpartum women who have been vaccinated or exposed to COVID-19: A systematic review", "authors": [ "Eighty Mardiyan Kurniawati", "Nur Anisah Rahmawati", "Nur Anisah Rahmawati" ], "abstract": "Background: Breast milk is a critical element in developing a baby's immunity through immune transfer. Antibodies are an essential unit of immunity against infection with the SARS-CoV-2 virus. This paper explores antibodies in breast milk in postpartum women who have been vaccinated or exposed to coronavirus disease 2019 (COVID-19). Duration of antibody appearance was studied to determine the adequate time in transferring antibodies by breastfeeding. Methods: Three databases, PubMed, Google Scholar, and ScienceDirect, were used as sources of articles. Inclusion criteria applied in selecting articles were prospective observational study or experimental design study in English, evaluating antibodies in breast milk, and conducted between 2019–2021. Article quality and risk of bias were assessed with Critical Appraisal Skills Programme (CASP). The data found were synthesized in a narrative manner. Results: This systematic review included 20 articles. A total of 306 postpartum women who were infected with COVID-19, 20 postpartum women who had viral symptoms and 495 postpartum women who had been vaccinated were studied. Immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies were found in the breast milk of infected and vaccinated postpartum women. SARS CoV-2 infection is associated with the presence of IgA dominant, whereas vaccination is related to the presence of IgG dominant. Antibodies persisted from day 10 of onset to 10 months in infected postpartum women and started from three days to six weeks in vaccinated postpartum women. Meta-analysis could not be carried out due to the variety of articles. Conclusions: Antibodies found in breast milk in infected and vaccinated postpartum women have different dominant types. Further research needs to be done regarding the mechanism of antibody transfer in breast milk, longer research duration and studies that directly examine the comparison of antibodies in breast milk in vaccinated and infected postpartum women. Registration: PROSPERO (CRD42022340859, 23 June 2022).", "keywords": [ "COVID-19", "maternal health", "neonatal health", "human milk", "antibody", "vaccine" ], "content": "Introduction\n\nSARS CoV-2 became a worldwide pandemic and caused changes in the phases of life, one of which was postpartum and neonatal periods.1 Coronavirus disease 2019 (COVID-19) infection during pregnancy, birth and puerperium is associated with a significant increase in morbidity and mortality in mothers and babies. They are considered people susceptible to infection compared to the general population.2 COVID-19-related postpartum deaths have been reported in Brazil, with an estimated 106 deaths in 2020.3 In addition, babies are also vulnerable to COVID-19 infection. The babies born to mothers infected with COVID-19 are more likely to be admitted to the neonatal unit.4 Cases of COVID-19 in children in previous studies were seen to be less frequent, less severe, and the mortality rate very low, but there is growing evidence that they are as susceptible as adults.5 Infants with severe respiratory failure and the prolonged clinical course associated with SARS-CoV-2 exposure may be due to extreme prematurity, immature lungs, and immunocompromised status.6\n\nBreast milk is the only nutrient needed in the first six months of life.7 The best source of nutrition and protection for babies from disease is breast milk.8 Breast milk can be an opportunity and hope for babies to achieve protection. The provision of a COVID-19 vaccine for newborns is not yet available. A recent study found that a COVID-19 vaccination regimen consisting of BNT162b2 was found to be safe, immunogenic, and efficacious in children aged five to 11 years.9 When compared with infants, postpartum mothers have the opportunity to get antibodies through vaccination. The COVID-19 vaccine is an effective way to prevent COVID-19 in breastfeeding people.10 In influenza outbreaks in previous years, influenza vaccines have been shown to increase serum antibodies and reduce disease severity in both mother and baby. Breastfeeding can protect you for at least six months because breast milk contains consistently high levels of actively produced anti-influenza immunoglobulin A (IgA). Infants with fever have fewer episodes of respiratory illness, which implies that breastfeeding may provide local mucosal protection.11 The risk of COVID-19 can be reduced through breastfeeding among children as has been documented for other infections compared to formula feeding.12\n\nThe risk of being exposed to the virus and providing nutrition is a dilemma, especially regarding the content of substances contained in breast milk. This is also exacerbated by the rise of fake news and misinformation on social media about the content of the SARS-CoV-2 virus in breast milk, causing breastfeeding issues during the pandemic.13 Mothers will be distraught and ask themselves whether the coronavirus can be transmitted through breast milk and what they can do to protect themselves and their babies.8 The World Health Organization (WHO) said breastfeeding does not need to be stopped during COVID-19 infection or after the mother’s vaccination.14 Research conducted by United Nations Children’s Fund (UNICEF) in five countries in South Asia found that less than 25% of interviewees understood that it was safe to continue breastfeeding. They preferred to give formula milk.15 Meta-analysis conducted in 2020 showed that the SARS-CoV-2 genome is generally not found in the breast milk of individuals infected with COVID-19.16\n\nAfter the virus content information has been clearly explained, the protection points become an essential part to know. Previous studies have found that there are antibodies in the breast milk of COVID-19-infected mothers, but the duration of the presence of these antibodies is unknown.17 Infected and vaccinated postpartum women allow the formation of an antibody against COVID-19, but the long-term impact on antibody composition and functional activity is unclear.18 Given this context, the purpose of this research is to determine the status of antibodies in breast milk following COVID-19 exposure and vaccination. After finding records of the presence of antibodies, the duration of the appearance of antibodies will also be studied. Knowledge about the duration of antibodies in breast milk determines the adequate time in transferring antibodies by breastfeeding. The current results can be used as a guideline for recommendations to continue breastfeeding and health promotions that emphasize the presence of antibodies in breast milk in vaccinated postpartum mothers.\n\n\nMethods\n\nThe status of antibodies in breast milk after exposure to COVID-19 and after vaccination and the duration of antibodies appearing in breast milk were explored by compiling a systematic review. The preparation of this report follows the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines.19,39 This systematic review is registered in PROSPERO (CRD42022340859, 23 June 2022). Data were collected in July-September 2021. English-language research conducted from 2020 to 2021 according to topics was searched in PubMed (RRID:SCR_004846), Google Scholar (RRID:SCR_008878), and Science Direct. The keywords used were a combination of Medical Subject Heading (MeSH) terms and relevant keywords in a different order: “breast milk”, “breast milk”, “COVID-19”, “antibody”, “immunoglobulin”, “vaccine”, “severe”, “acute respiratory syndrome coronavirus 2”, “COVID19”, “coronavirus disease 2019”, “SARS-CoV-2”. In the articles found, the researcher also examined the literature in the bibliography, including manuscripts that were not captured in the electronic literature search.\n\nThe inclusion criteria applied in selecting articles were prospective observational study or experimental design study in English, evaluating antibodies in breast milk, and the study was conducted during the COVID-19 pandemic between 2019 and 2021. The exclusion criteria for this study were case reports, animal studies, letters to editors, study reviews, abstracts without full text. Manuscripts that only discussed breast milk in healthy postpartum mothers were not included.\n\nTwo authors (EMK and NAR) performed title and/or abstract screening independently of the included articles using standard Microsoft Excel (RRID:SCR_016137) forms. The data obtained were combined in one folder and then an assessment was carried out. Each author analyzed all existing manuscripts, and then the results were compared with each other. A third external collaborator was consulted (Hari Paraton) to address disagreements in consensus.\n\nThe outcomes sought from this study were the presence of antibodies and the duration of the appearance of antibodies in breast milk. The antibodies studied were IgG and IgA against COVID-19. The tool to detect the presence of this antibody used ELISA or a similar working tool. The researcher realized that not all manuscripts had explanations for these two antibody levels, so studies that only discussed one type of antibody were also included in the systematic review. The researcher also realized that not all studies that discussed antibodies also explained the duration of the appearance, so based on the consideration of the limited number of articles, the researcher discussed the data found from existing studies. Unclear information was included in the exclusion criteria.\n\nResearchers conducted a risk of bias assessment study with the help of critical appraisal tools. The Critical Appraisal Skills Programme (CASP) was used to assess the formal article by two independent research team members, EMK and NAR. Questions are designed to help systematically review a cohort study. The use of CASP is based on the 1994 JAMA ‘Users’ guides to the medical literature, which is used for both randomized controlled trials and systematic reviews. This checklist was adapted from Guyatt GH, Sackett DL, and Cook DJ and is used by health care practitioners.20 CASP results are concluded into the category of moderate overall quality and low overall quality.21 The results of the analysis are presented in Table 1.\n\nDue to the limited number of manuscripts, a meta-analysis could not be completed. The effect that was measured in this study was the number of respondents’ breast milk presentations where antibodies were found in breast milk. The obtained durations were also combined descriptively.\n\nThe study selection process was through a review of the inclusion and exclusion criteria. As suggested in a systematic review of the literature, the analysis in this study was based on the findings of each study. The steps started from extracting the relevant results, sorting, and examining them to identify sub-themes and themes. The type of synthesis used is in the form of narrative synthesis. Narrative synthesis was chosen because it allowed researchers to gather insight into the antibody content in breast milk and we did not perform this meta-analysis. The data are arranged in Tables 2 and 3. Table 2 contains the antibody content in breast milk in mothers exposed to COVID-19 and is written in a systematic table including (1) authors, year, country, (2) research time, (3) study type, (4) COVID-19 confirmation, (5) control group, (6) sampling time, (7) number of infected mothers, (8) antibody test, (9) number of mothers showing antibodies, and (10) duration time. Table 3 contains antibody content in breast milk in mothers vaccinated against COVID-19 and is written in a systematic table including (1) authors, year, country, (2) research time, (3) study type and sample timing, (4) vaccine type, (5) number of vaccinated women and the dose given, (6) antibody test, (7) antibody status, and (10) duration time. Heterogeneity in the data was explored including duration of baseline antibody measurement and population characteristics.\n\nThe researcher was unable to review the funnel plot in this case due to the limitations of the manuscript so that the risk assessment of bias from missing results in the synthesis was carried out using an emerging risk approach and was reported as a research constraint.\n\nThe main domain used to assess the certainty of evidence was the risk of bias through critical appraisal and the inconsistency of the results of the research included in the review.\n\n\nResults\n\nIn searching the database, 1,465 abstracts were found from searches with relevant keywords. The authors then screened results for possible inclusion. The findings of 935 articles were excluded from the eligibility list. The articles did not meet the criteria, including the type of manuscript, the language used, and the topics discussed under the proposed title. After this stage, the researcher tried to re-examine the assessment results, but the manuscript finally entered the exclusion criteria. After screening, 30 full-text articles were selected and examined in detail to determine eligibility. Furthermore, 20 articles were determined that met the requirements. There were eight studies on the antibody status of infected breastfeeding mothers and 12 studies on antibody status of vaccinated breastfeeding mothers included. Figure 1 shows the study selection flowchart. The quality of the research is in the moderate category and is arranged in Table 1.\n\nCOVID-19, coronavirus disease 2019.\n\nA total of 306 breastfeeding mothers who were positive for COVID-19 and 20 of them who had viral symptoms and had a very high likelihood that they had been exposed to COVID-19 were studied to determine antibody status in breast milk. Samples were taken within 48 hours after delivery to six months after infection. Table 2 describes antibody status in mothers who had been infected with COVID-19. The samples included in this review are breastfeeding mothers with positive PCR confirmation and mothers who had a very high likelihood that they had been exposed to COVID-19 when viewed from the symptoms they had. Almost all studies used controls, namely comparisons with healthy breastfeeding mothers and milk samples taken during the pre-pandemic period. One study did not include controls, namely a longitudinal study. Detection of all antibodies was performed using ELISA. Most studies found that breast milk contains IgA, immunoglobulin G (IgG), and immunoglobulin M (IgM). IgA was the dominant antibody found, while IgG and IgM were not always found in individuals. However, infected mothers also did not always have IgA antibodies against COVaID-19, which was found in a study by Juncker et al. (2021), who found that 59% of mothers had these antibodies.22\n\nThe characteristics of the study on antibody status in the breast milk of vaccinated mothers can be seen in Table 3. A total of 495 breastfeeding mothers who had been vaccinated, received the first and second doses of various types of vaccines and had never had COVID-19 were analyzed. IgG and IgA antibodies were found in the breast milk of vaccinated mothers. No studies showed IgM data. Administration of the second dose of the vaccine was associated with an increase in IgG in breast milk. Despite evidence of increased antibodies, anti-SARS-CoV-2 IgG antibodies were not detected in the plasma of infants whose mothers were vaccinated during breastfeeding. IgG levels were higher than IgA levels in the breast milk of vaccinated mothers.\n\nThe duration of antibody time associated with PCR confirmation or symptoms, breast milk testing, and vaccine administration is shown in Figure 1. In mothers infected with COVID-19, the appearance of antibodies was detected on day 10 and could persist for up to 10 months. IgA levels are known to decrease over time, while IgG is relatively stable. In vaccinated mothers, antibody status differed between mothers vaccinated with dose one and dose two. Most studies found the presence of antibodies after the first dose, although in small amounts. Early appearance could be detected three days after vaccination up to six weeks in both dose one and dose two of vaccination. The highest levels and immune response were found about four weeks after vaccination, but at the second dose, the antibody appeared between days four and 10. After that, there may be a chance to experience a decline before the sixth week, especially on day 15 or day 43 ± 4. These studies stopped examining antibodies at the sixth week. Therefore, the data provided are limited to detection at the sixth week. The results of existing studies indicate that it is not known whether, after six weeks, there is still a decrease or whether it persists at a certain point.\n\nNot all studies discussed the comparison of antibodies in various types of vaccines. There were two studies that explored this. Research conducted by Lechosa-Muñiz et al. (2021) concluded that BNT162b2 or mRNA-1273 vaccinated mothers had higher levels of IgG antibodies compared to ChAdOx1-S (p < 0.001 and p = 0.001), respectively.23 Mean values in serum from mothers vaccinated with BNT162b2 or mRNA-1273 vs. ChAdOx1-S (0.12, 0.16, and 0.02, respectively; p < 0.001) on IgA antibodies. Regardless of the commercial vaccine administered, all vaccinated breastfeeding mothers had serum anti-S1 IgG antibodies in response to vaccination against SARS-CoV-2.23 Research conducted by Selma-Royo et al. (2021) found that mothers vaccinated with Moderna and BioNTech/Pfizer after the first dose had a higher increase in breast milk anti-SARS-CoV-2 IgG than Oxford/AstraZeneca (p < 0.0001).24 Anti-SARS-CoV-2 IgA levels were higher in Moderna than in the Oxford/AstraZeneca (p < 0.0001) and BioNTech/Pfizer groups (p = 0.002). After the second dose, no difference was observed between the two mRNA-based vaccines. Moreover, after the second dose, IgG levels reached a higher level than the first dose, whereas IgA levels did not increase further.24 Gray et al. (2021) found that a booster dose of the vaccine increases SARS CoV-2 IgG, but not IgA, in maternal blood and breast milk.25\n\n\nDiscussion\n\nThe antibody status in the breast milk of mothers who have been exposed to COVID-19 or are highly suspected and those who have been vaccinated against COVID-19 differs. The dominant antibody in mothers infected with COVID-19 is IgA, while the predominant antibody in vaccinated mothers is IgG, according to Young et al. (2021).18 Vaccination is associated with an increase in dominant IgG and IgA antibodies after direct viral infection. This varies greatly. Breast milk from both groups showed neutralizing activity against live SARS-CoV-2 virus, which could be attributed to SARS-CoV-2 IgA and IgG antibodies.18 The dominant IgA response in breast milk can be found according to previous studies, namely the result of natural infection.26\n\nAntibody transfer through breast milk is an evolutionary strategy for enhancing immunity early in life.27 Research conducted by Pullen et al. (2021) in which they applied a serologic system to characterize SARS-CoV-2 specific antibodies in maternal serum and breast milk found a preferential transfer of antibodies capable of causing neutrophil phagocytosis and neutralization. Distinct SARS-CoV-2-specific antibody response was observed in serum and breast milk from nursing individuals previously infected with SARS-CoV-2, with a predominant transfer of IgA and IgM into breast milk.27 IgA is the most essential class of Ig provided by breast milk to infants because it acts in the intestines while the secretory IgA (SIgA) produced by infants is still in development.28 The presence of IgM in some samples suggests the possibility that breast milk may have a protective effect on the newborn.29\n\nAlthough IgGs are present in breast milk, they are functionally attenuated.27 Emerging data from vaccinated pregnant and lactating women suggest that vaccine-induced transfer may be altered due to unusually high levels of IgG antibody induced by mRNA vaccines approved by the current Emergency Use Authorization.25 It gives the baby strong IgA and IgG antibodies and may increase immunity compared to natural infection.27 Nevertheless, the IgG transfer scheme still needs to be studied further because different studies can find different results. A study conducted by Golan et al. (2021) found that no IgG was found in the plasma of infants whose mothers were vaccinated during lactation.30 Although high levels of IgG were found in breast milk, these antibodies may not be transferred effectively to the baby. IgA antibodies produced after vaccination with the Pfizer/BioNTech vaccine resist the gastric phase but are degraded during the intestinal phase of the infant’s digestion. By contrast, IgG is more susceptible to degradation in both digestive phases.31 The results of another study found that maternal SARS-CoV-2 IgG was efficiently transferred across the placenta when infection occurs more than two months before delivery. Passive immunity inherited from the mother can last in infants for up to six months. Neonates can mount a strong antibody response to perinatal SARS-CoV-2 infection.32 Antibodies shown from breastfeeding may have a protective effect on the recipient infant, provided that the infant has not increased its immune response to infection.33\n\nStudies showing the duration of antibody persistence in breast milk are limited. Antibodies can be detected in mothers who are breastfeeding and infected with COVID-19 as early as day 10 and can last for up to 10 months. IgA levels are known to decrease over time, while IgG is relatively stable. In lactating mothers who were vaccinated, the trial was only up to six weeks in duration. Antibodies have been detected after the first dose of vaccination for three days and can last up to six weeks. Before six weeks, it can decrease. It is not known whether the antibody status will still decrease or persist at a certain point. Individual research by Young et al. (2021) showed that IgG began to decline by 90 days after the second vaccine dose.18 This suggests that further research is needed to investigate the exact duration of antibodies in breast milk after vaccination.\n\nThe type of vaccine is related to the number of antibodies detected in breast milk even though IgG status is more dominant than IgA and IgM. Post-vaccination IgG levels reached levels similar to those of terminally ill COVID-19 patients and demonstrated a decreased breadth of antibody responses targeting the endemic coronavirus.34 Another study conducted by Dashdorj et al. (2021) found that responses from high to low were Pfizer/BioNTech, AstraZeneca, Sputnik V and Sinopharm, respectively.35 These different responses also allow different amounts of antibodies to be transferred into breast milk.\n\nBased on the results of the opportunity for antibody transfer, breastfeeding is highly recommended for mothers infected with COVID-19 and mothers who have been vaccinated. Research conducted by Verd et al. (2021) found that in a sample of children visiting emergency services with potential symptoms of COVID-19, a higher prevalence of positive SARS-CoV-2 reverse transcriptase (RT)-PCR test results among those who were formula-fed exclusively compared to those who have been breastfed. Breastfeeding can reduce the risk of exposure to COVID-19 and other infections in children, compared to formula feeding.12 It is vital to continue to breastfeed according to the available time duration. Therefore, women with confirmed COVID-19 must adhere to standard precautions for contact with breastfeeding.36 The need to encourage breastfeeding may be justified, at least during the first six months of life, when the infant’s secretory IgA production is insignificant.37 Apart from direct breastfeeding, every mother has the right to choose to breastfeed her baby and health workers need to help ensure good handwashing practices before and after expressing the milk.38\n\nMost of these studies only involve a small number of samples, and they did not find a mechanism for antibody transfer during breastfeeding. We recommend that future research explores antibody testing with a more extended research duration (more than six weeks) in vaccinated postpartum women. In order to ensure that the reported time is more accurate, multiple individual studies that directly discuss the comparison of antibodies in vaccinated mothers with mothers exposed to COVID-19 and the assessment of antibody transfer mechanism by breastfeeding may be useful. In addition, the researcher was unable to compile a meta-analysis because it included various types of academic papers.\n\nThe strength of this study is that there have not been many studies that have reviewed the comparison of antibody types and duration of antibodies in infected and vaccinated postpartum women. The results obtained can support evidence-based health promotion to support continued breastfeeding during the pandemic and allow for the right time to support antibody transfer.\n\n\nConclusions\n\nIgA and IgG antibodies were found in the breast milk of infected and vaccinated postpartum women. Infection with SARS CoV-2 is related with the presence of IgA, but vaccination is associated with increased IgG. Antibody levels persisted from day 10 of onset to 10 months in infected breastfeeding mothers and start from three days to six weeks in vaccinated breastfeeding mothers. Antibodies produced in breast milk in infected and vaccinated postpartum women have different dominant types. Further research needs to be done regarding the mechanism of antibody transfer in breast milk, longer research duration and studies that directly examine the comparison of antibodies in breast milk in vaccinated and infected postpartum women.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReporting guidelines\n\nZenodo: PRISMA checklist for ‘Records of antibodies in breast milk in postpartum women who have been vaccinated or exposed to COVID-19: A systematic review. https://doi.org/10.5281/zenodo.6785317.39\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nWe would like to thank Hari Paraton as an external contributor to this paper who was consulted to address disagreements in consensus during the data collection process.\n\n\nReferences\n\nKotlar B, Gerson E, Petrillo S, et al.: The impact of the COVID-19 pandemic on maternal and perinatal health: a scoping review. Reprod. Health. 2021; 18(1): 10. PubMed Abstract \| Publisher Full Text\n\nVillar J, Ariff S, Gunier RB, et al.: Maternal and Neonatal Morbidity and Mortality Among Pregnant Women With and Without COVID-19 Infection: The INTERCOVID Multinational Cohort Study. JAMA Pediatr. 2021 Aug 1; 175(8): 817–826. PubMed Abstract \| Publisher Full Text\n\nGurzenda S, Castro MC: COVID-19 poses alarming pregnancy and postpartum mortality risk in Brazil. EClinicalMedicine. 2021; 36: 100917. PubMed Abstract \| Publisher Full Text\n\nAllotey J, Stallings E, Bonet M, et al.: Clinical manifestations, risk factors, and maternal and perinatal outcomes of coronavirus disease 2019 in pregnancy: Living systematic review and meta-analysis. BMJ. 2020; 41(September): 81–82. Publisher Full Text\n\nLiguoro I, Pilotto C, Bonanni M, et al.: SARS-COV-2 infection in children and newborns: a systematic review. Eur. J. Pediatr. 2020/05/18. 2020 Jul; 179(7): 1029–1046. PubMed Abstract \| Publisher Full Text\n\nKumar VHS, Prasath A, Blanco C, et al.: Respiratory Failure in an Extremely Premature Neonate with COVID-19. Child (Basel, Switzerland). 2021 Jun 4; 8(6): 477. Publisher Full Text Reference Source\n\nSchindler-Ruwisch J, Phillips KE: Breastfeeding During a Pandemic: The Influence of COVID-19 on Lactation Services in the Northeastern United States. J. Hum. Lact. 2021; 37(2): 260–268. PubMed Abstract \| Publisher Full Text\n\nUNICEF: Breastfeeding during the COVID-19 pandemic Tips on keeping your baby healthy and safe.2021.Reference Source\n\nWalter EB, Talaat KR, Sabharwal C, et al.: Evaluation of the BNT162b2 Covid-19 Vaccine in Children 5 to 11 Years of Age. N. Engl. J. Med. 2022; 386(1): 35–46. PubMed Abstract \| Publisher Full Text\n\nCDC: COVID-19 Vaccines While Pregnant or Breastfeeding.2021.Reference Source\n\nSchlaudecker EP, Steinhoff MC, Omer SB, et al.: IgA and neutralizing antibodies to influenza a virus in human milk: a randomized trial of antenatal influenza immunization. PLoS One. 2013 Aug 14; 8(8): e70867–e70867. PubMed Abstract\n\nVerd S, Ramakers J, Vinuela I, et al.: Does breastfeeding protect children from COVID-19? An observational study from pediatric services in Majorca, Spain. Int. Breastfeed. J. 2021; 16(1): 83. PubMed Abstract \| Publisher 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): 460–471. Publisher Full Text\n\nWHO: WHO recommends continuing breastfeeding during COVID-19 infection and after vaccination.2021.Reference Source\n\nUNICEF: Misinformation and formula milk donations are threatening breastfeeding during COVID-19 Breast milk is a baby’s ‘first vaccine”.’2020.Reference Source\n\nLow JM, Low YW, Zhong Y, et al.: Titres and neutralising capacity of SARS-CoV-2-specific antibodies in human milk: a systematic review. Arch. Dis. Child - Fetal Neonatal Ed. 2021 Jul 12. fetalneonatal-2021-322156.Reference Source\n\nUNICEF: Breastfeeding Safely During The COVID-19 Pandemic.2021.\n\nYoung BE, Seppo AE, Diaz N, et al.: Association of Human Milk Antibody Induction, Persistence, and Neutralizing Capacity With SARS-CoV-2 Infection vs mRNA. Vaccination. 2021; 176: 159–159. Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009 Jul 21; 6(7): e1000097. PubMed Abstract \| Publisher Full Text\n\nCritical Appraisal Skills Programme (CASP): CASP Checklist: Cohort Study. Casp UK.2018; (2018): 7.Reference Source\n\nLong HA, French DP, Brooks JM: Optimising the value of the critical appraisal skills programme (CASP) tool for quality appraisal in qualitative evidence synthesis. Res. Methods Med. Heal. Sci. 2020 Aug 6; 1(1): 31–42. Publisher Full Text\n\nJuncker HG, Romijn M, Loth VN, et al.: Antibodies Against SARS-CoV-2 in Human Milk: Milk Conversion Rates in the Netherlands. J. Hum. Lact. 2021; 37: 469–476. Publisher Full Text\n\nLechosa-Muñiz C, Paz-Zulueta M, Mendez-Legaza JM, et al.: Induction of sars-cov-2-specific igg and iga in serum and milk with different sars-cov-2 vaccines in breastfeeding women: A cross-sectional study in northern spain. Int. J. Environ. Res. Public Health. 2021; 18(16): 1–12.\n\nSelma-Royo M, Bauerl C, Mena-Tudela D, et al.: Anti-Sars-Cov-2 IgA And IgG In Human Milk After Vaccination Is Dependent On Vaccine Type And Previous Sars-Cov-2 Exposure A Longitudinal Study. medRxiv. 2021. 2021.05.20.21257512. Publisher Full Text\n\nGray KJ, Bordt EA, Atyeo C, et al.: Coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study. Am. J. Obstet. Gynecol. 2021; 225(3): 303.e1–303.e17. PubMed Abstract \| Publisher Full Text\n\nvan Keulen BJ , Romijn M, Bondt A, et al.: Human milk from previously covid-19-infected mothers: The effect of pasteurization on specific antibodies and neutralization capacity. Nutrients. 2021; 13(5). Publisher Full Text\n\nPullen KM, Atyeo C, Collier ARY, et al.: Selective functional antibody transfer into the breastmilk after SARS-CoV-2 infection. Cell Rep. 2021; 37(6): 109959. PubMed Abstract \| Publisher Full Text\n\nRio-Aige K, Azagra-Boronat I, Castell M, et al.: The Breast Milk Immunoglobulinome. Nutrients. 2021; 13. PubMed Abstract \| Publisher Full Text\n\nPeng S, Zhu H, Yang L, et al.: A study of breastfeeding practices, SARS-CoV-2 and its antibodies in the breast milk of mothers confirmed with COVID-19. Lancet Reg. Heal. - West Pacific. 2020; 4: 100045. PubMed Abstract \| Publisher Full Text Reference Source\n\nGolan Y, Prahl M, Cassidy AG, et al.: COVID-19 mRNA Vaccination in Lactation: Assessment of adverse effects and transfer of anti-SARS-CoV2 antibodies from mother to child. medRxiv Prepr. Serv. Heal. Sci. 2021. Reference Source\n\nPieri M, Nicolaidou V, Paphiti I, et al.: Survival of vaccine-induced human milk SARS-CoV-2 IgG and IgA immunoglobulins across simulated human infant gastrointestinal digestion. medRxiv. 2021. 2021.06.17.21259021. Publisher Full Text\n\nSong D, Prahl M, Gaw SL, et al.: Passive and active immunity in infants born to mothers with SARS-CoV-2 infection during pregnancy: prospective cohort study. BMJ Open. 2021 Jul 1; 11(7): e053036. PubMed Abstract \| Publisher Full Text Reference Source\n\nFavara DM, Ceron-Gutierrez ML, Carnell GW, et al.: Detection of breastmilk antibodies targeting SARS-CoV-2 nucleocapsid, spike and receptor-binding-domain antigens. Emerg. Microbes Infect. 2020 Dec; 9(1): 2728–2731. PubMed Abstract\n\nRöltgen K, Nielsen SCA, Arunachalam PS, et al.: mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition. medRxiv Prepr. Serv. Heal. Sci. 2021 Apr 7. 2021.04.05.21254952.Reference Source\n\nDashdorj NJ, Wirz OF, Röltgen K, et al.: Direct comparison of antibody responses to four SARS-CoV-2 vaccines in Mongolia. Cell Host Microbe. 2021; 29(12): 1738–1743.e4. PubMed Abstract \| Publisher Full Text Reference Source\n\nPramana C, Suwantoro J, Sumarni N, et al.: Breastfeeding in postpartum women infected with COVID-19. Int. J. Pharm. Res. 2020; 12(14): 1857–1862.\n\nPalmeira P, Carneiro-Sampaio M: Immunology of breast milk. Rev. Assoc. Med. Bras. 2016 Sep 1; 62: 584–593. Publisher Full Text\n\nMarinelli KA, Lawrence RM: Safe Handling of Containers of Expressed Human Milk in all Settings During the SARS-CoV-2 (COVID-19) Pandemic. J. Hum. Lact. 2020; 36: 498–501. Publisher Full Text\n\nKurniawati EM: Records of antibodies in breast milk in postpartum women who have been vaccinated or exposed to COVID-19: A systematic review. [Dataset]. Zenodo.2022. Publisher Full Text\n\nJuncker HG, Romijn M, Loth VN, et al.: Human Milk Antibodies Against SARS-CoV-2: A Longitudinal Follow-Up Study. J. Hum. Lact. 2021; 37: 485–491. Publisher Full Text\n\nNarayanaswamy V, Pentecost B, Alfandari D, et al.: Humoral and Cell-Mediated Immune Response in Colostrum from Women Diagnosed Positive for SARS-CoV-2. Breastfeed. Med. 2021; 16: 987–994. Publisher Full Text\n\nBäuerl C, Randazzo W, Sánchez G, et al.: SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain. Arch. Dis. Child - Fetal Neonatal Ed. 2021. fetalneonatal-2021-322463.\n\nDuncombe CJ, Mcculloch DJ, Shuey KD, et al.: Dynamics of breast milk antibody titer in the six months following SARS-CoV-2 infection. J. Clin. Virol. 2021; 142(January): 104916. PubMed Abstract \| Publisher Full Text\n\nPace RM, Williams JE, Järvinen KM, et al.: Characterization of sars-cov-2 rna, antibodies, and neutralizing capacity in milk produced by women with covid-19. MBio. 2021; 12(1): 1–11. Publisher Full Text\n\nFox A, Marino J, Amanat F, et al.: Evidence of a significant secretory-IgA-dominant SARS-CoV-2 immune response in human milk following recovery from COVID-19. medRxiv. 2020.\n\nDemers-Mathieu V, Dapra C, Mathijssen G, et al.: Human milk antibodies against s1 and s2 subunits from sars-cov-2, hcov-oc43, and hcov-229e in mothers with a confirmed covid-19 pcr, viral symptoms, and unexposed mothers. Int. J. Mol. Sci. 2021; 22(4): 1–13.\n\nLow JM, Gu Y, Ng MSF, et al.: Codominant IgG and IgA expression with minimal vaccine mRNA in milk of BNT162b2 vaccinees. npj Vaccines. 2021; 6(1): 105. PubMed Abstract \| Publisher Full Text\n\nJakuszko K, Kościelska-Kasprzak K, Żabińska M, et al.: Immune response to vaccination against covid-19 in breastfeeding health workers. Vaccines. 2021; 9(6): 1–10. Publisher Full Text\n\nGuida M, Terracciano D, Cennamo M, et al.: COVID-19 vaccine mRNABNT162b2 elicits human antibody response in milk of breastfeeding women. Vaccines. 2021; 9(7): 3–9. Publisher Full Text\n\nRamìreza DSR, Pérez MML, Pérez MC, et al.: SARS-CoV-2 Antibodies in Breast Milk After Vaccination. Pediatrics. 2021; 148: e2021052286. Publisher Full Text\n\nPerl SH, Uzan-Yulzari A, Klainer H, et al.: SARS-CoV-2–Specific Antibodies in Breast Milk After COVID-19 Vaccination of Breastfeeding Women. JAMA. 2021 May 18; 325(19): 2013–2014. PubMed Abstract \| Publisher Full Text\n\nValcarce V, Stafford LS, Neu J, et al.: Detection of SARS-CoV-2-Specific IgA in the Human Milk of COVID-19 Vaccinated Lactating Health Care Workers. Breastfeed. Med. 2021; XX(Xx): 1–6.\n\nJuncker HG, Mulleners SJ, van Gils MJ , et al.: The Levels of SARS-CoV-2 Specific Antibodies in Human Milk Following Vaccination. J. Hum. Lact. 2021; 37(3): 477–484. PubMed Abstract \| Publisher Full Text\n\nRosenberg-Friedman M, Kigel A, Bahar Y, et al.: BNT162b2 mRNA vaccine elicited antibody response in blood and milk of breastfeeding women. Nat. Commun. 2021; 12(1): 1–7." }	[ { "id": "144205", "date": "18 Aug 2022", "name": "Nicole Cacho", "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\nKurniawati and Rahmawati’s interesting manuscript “Records of antibodies in breast milk in postpartum women who have been vaccinated or exposed to COVID-19: A systematic review” summarizes the various manuscripts on breast milk antibodies following COVID-19 exposures and vaccinations. It is obvious the authors worked hard to review the manuscripts and compose the manuscript.\nThe article highlighted the specific antibody findings of trends of which type of antibodies were more prevalent after vaccination compared to COVID-19 infection. This extensive work on reviewing 20 cohort studies has highlighted trends in antibodies and laid groundwork for future systematic reviews on studies with longer follow up and possible meta-analysis. Unfortunately, the differences in methods and lack of homogeneity made it impossible to perform a meta-analysis. This work is an important addition to the literature on breast milk immunity in the setting of a global pandemic with COVID-19.\nMajor Revisions include the following:\nOverall, the grammar needs extensive editing.\nThis is especially needed in the Introduction section. Many of the sentences below are confusing to the extent that I unsure of the meaning the author is trying to convey.\nIntroduction section with confusing sentences (page numbers refer to the pdf version of the manuscript): Page 1, 1st paragraph, First line: …and caused changes in the phases of life. In what ways? Page 1, 1st paragraph, 3rd sentence: Consider changing to New mothers and their infants are more susceptible to infection compared to the general population. Page 1, 2nd paragraph, “Breast milk is the only nutrient needed in the first 6 months of life.” Did you mean only nutrition? Page 1, 2nd paragraph, “Breast milk can be an opportunity and hope for babies to achieve protection”. Would take out hope to make it more scientific. Page 1, 2nd paragraph, “Breastfeeding can protect “you” for at least six months because breast milk contains…” Vague you is confusing. Instead would say “Breastfeeding can provide protection for at least the first 6 months because breast milk contains… Page 1, 3rd paragraph, The risk of being exposed to the virus and providing nutrition is a dilemma… What does this mean? Dilemma to whom? To the mom, or for the baby? Why? Page 1, 3rd paragraph, would not use “fake news” which is slang, instead keep the word misinformation.\n\nDiscussion section sentences which need clarification Page 11, 1st discussion paragraph, “This varies greatly”, What varies greatly? Page 11, 1st discussion paragraph, “Breast milk from both groups showed neutralizing activity…” Which groups? Page 11, 2nd discussion paragraph, “IgA is the most essential glass of Ig provided by breast milk…produced by infants is still in development.” What is still in development? \"The presence of IgM in some samples (breast milk) suggests the possibility that breast milk may have a protective effect on the newborn.” In what way in the context of this systematic review?\nPage 11, 3rd paragraph, 3rd sentence, “It gives the baby strong IgA and IgG antibodies and may increase immunity comparted to natural infection.” What is “it”? mRNA vaccines? If so, say these vaccines or mRNA vaccines.\n\nPage 12 of discussion first sentence, “Needs to be studied further because different studies can find different results.” This sentence is too vague. Do you mean that studies report conflicting results? If so, say that.\n\nPage 12, 3rd paragraph , Dashdorj et al. 2021, which antibody?\n\nBe consistent throughout the manuscript and tables when describing the population.\nDetermining the difference between exposed to COVID-19 or infected with COVID-19 (or even suspected infection with COVID-19). This point is important as many people that are exposed may not know they are exposed and if asymptomatic will likely not get tested. Thus, simply exposed does not seem like the description that describes the majority of the population in these 20 studies. Be consistent in the terms of infected or exposed. For example, the title of Table 2 is “Characteristics of research on mothers with COVID-19 and antibody status in breast milk” but on Page 4 under the section Data abstraction and synthesis, “Table 2 is antibody contact in mothers exposed to COVID-19” but later on Page 10 under Antibody status in breast milk from infected other.“ Table 2 disuses antibody status in mothers who had been infected with COVID-19. Based on the majority of the studies confirming COVID-19 with PCR (with the exception of 1 study by Bauerl et al. 2021 which had CPR and/or after COVID-19 infection), I would advise changing the title of Table 2 to “Characteristics of research on mothers infected with COVID-19 and antibody status in breast milk”. Furthermore, I would not refer to Table 2 in the text as mothers who had been exposed.\n\nRecommend removing or modifying certain points that are not necessary to the flow of the manuscript.\n\nPage 1, 2nd paragraph, “Infants with fever have fever episodes of respiratory illness, which implies that breastfeeding may provide local mucosal protection?\" I’m not sure what that means. Does this mean all infants with fever, or with COVID-19? If it’s a general statement of all infants with fever of unknown cause, it does not add to the paragraph. If it’s meaning infants with COVID-19 with fever, then say that.\nPage 12, 4th paragraph, I would remove the sentence “Therefore, women with confirmed COVID-19 must adhere to standard precautions for contact with breastfeeding.\" Contact precautions were not previously discussed and may be confusing or send unclear messages. If it is strongly desired to keep this sentence, would change must to “should be advised” and also cite WHO, CDC and AAP recommendations for precautions.\nPage 12, 4th paragraph, I would update the sentence “The need to encourage breastfeeding may be justified at least during the first 6 moths of life, when the infant’s secretory IgA production is insignificant.” There are clear and updated recommendations on the duration of breastfeeding from American Academy of Pediatrics, UNICEF and WHO: which all say exclusive breastfeeding for first 6 months and then up to 2 years of age. You could then highlight the importance of the first 6 month based on infant’s low production of sIgA after discussion the general breastfeeding duration recommendations.\n\nWHO and UNICEF recommend: - early initiation of breastfeeding within 1 hour of birth; - exclusive breastfeeding for the first 6 months of life; and - introduction of nutritionally-adequate and safe complementary (solid) foods at 6 months together with continued breastfeeding up to 2 years of age or beyond. (9 June 2021) https://www.who.int/news-room/fact-sheets/detail/infant-and-young-child-feeding\nAAP supports continued breastfeeding, along with appropriate complementary foods introduced at about 6 months, as long as mutually desired by mother and child for 2 years or beyond. These recommendations are consistent with those of the World Health Organization (WHO). (See below for citation)\nJoan Younger Meek, Lawrence Noble, Section on Breastfeeding; Policy Statement: Breastfeeding and the Use of Human Milk. Pediatrics July 2022; 150 (1): e2022057988. 10.1542/peds.2022-057988\n\nImportant to use language that qualifies interpretations of studies that suggest findings but not strongly. For instance, on Page 12, 4th paragraph, “Breastfeeding can reduce the risk of exposure to COVID-19 and other infections in children when compared to formula feeding (reference 12 by Verd et al. 2021).\" This is too strong of a statement since the cohort was retrospectively reviewed in a secondary analysis and used “Any breastfeeding” vs. exclusive formula feeding and these were in children up to 13 years of age. The conclusion section uses the word suggest that there is protection with breastfeeding. Thus, I recommend changing the work “can” reduce to “may” reduce.\n\nImportant point to consider - Mechanism of antibody transfer in the breast milk. I am uncertain what the meaning of this statement is. As per World Health Organization, the presence of IgA in breast milk is one of the ways in which breastfeeding protects infants against infection and death. This appears to be a big part of the conclusion section for future direction but it is not clear that it is based on the results presented.\n\nTables\nTable 1: The question of How precise are the results? Is not a yes/ no question. Per CASP checklist for cohort study the included HINT was Look for range of confidence interval. I would also include p values if given. If not available, list “no data available” for that particular study. This is important for the statistical analysis portion. After you can compare and contrast studies within each group of vaccinated or infected mothers.\n\nTable 2: To make it more consistent for no. of infected mothers, just include the numerical value, (two are in words).\nTable 2: Bauerl et al. sampling time is listed as before and after. How many days or weeks? Also how is infection defined in this study, if not by PCR?\nTable 2: last entry Demers-Mathieu et al lists sampling time as 16-84 days. What is that in relation to?\nTable 2 Heading Duration time: What is this the duration of? Follow up with patients?\nTable 3 Heading Time: of what? Timing after vaccination when antibody levels are measured?\nTable 3: Perl et al: which antibodies under “Antibody status”?\nIt would be informative to see an addition of antibody breakdown by type added to Table 3.\n\nFigures\nFigure 1 is the Flow chart of the study selection but Figure 1 is mentioned under Results subheading Time duration of antibodies appearing and is described as showing “Duration of antibody time associated with PCR confirmation or symptoms, breast milk testing, and vaccine administration. Was this supposed to be labeled as Figure 2. If so, I didn’t see a Figure 2.\nFigure 1 Flowchart is confusing because the numbers do not add up as they are explained. It appears that from the 1465, 500 were eliminated to leave 965. Then another 935 were eliminated to leave 30. Next 10 were eliminated to ultimately leave 20 studies. Each elimination should be explained clearly and the numbers should match up. Minor point: From 30 to 20 in the last 2 boxes in the flowchart reports excluded explained 11 that were eliminated but 30-11=19, not 20. Please revise this flowchart using an example from another systematic review.\n\nSmall revision points:\nIn methods on keyword search, it seems that “breast milk” and “COVID-19” were typed twice as an error. Please fix this. These are details of the methods and analysis to allow replication by others.\nWhat is meant by “unclear information was included in the exclusion criteria”? Is this for a few of the studies that were not included or is this describing the majority of the 20 studies that were included?\nWhat is a funnel plot? If you mention it, you may want to explain it.\n\nPage 10, last sentence, typo: COVaID-19 instead of COVID-19.\nPage 11, Results section, Heading Antibody status in breast milk from vaccinated, second to last sentence, Serum levels in infants appears to only be included in one study, so please provide the reference for that study as a superscript and also mention it was only one of the 20 studies. Also, how many infants had serum levels? Not many mothers would want you to poke their babies to get a blood draw.\n\nPage 11, Results section, heading time duration of antibodies appearing, Last sentence is an interpretation of results thus, belongs in discussion section (which you already included on page 12, 2nd paragraph), so you can delete this sentence.\n\nLimitations and recommendations section: instead of the last sentence, “…because it included various types of academic papers.” Consider saying lack of homogeneity.\n\nWhy do Pace et al. and Fox et al. not fit with other available evidence? This should be explained in the discussion section. Personally, from Table 2 the Demers-Mathieu et all paper has all 26 patients with all antibodies in milk including IgA, IgG and IgM. I don’t believe those results. Do you?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "8665", "date": "30 Aug 2022", "name": "eighty mardiyan kurniawati", "role": "Author Response", "response": "Dear reviewers, Thank you for being willing to review our manuscript and give suggestions to improve its quality. We've tried to revise it. I hope you are satisfied with our revision. Thank you for your time. Best regards" } ] } ]	1	https://f1000research.com/articles/11-785
https://f1000research.com/articles/12-357/v1	31 Mar 23	{ "type": "Method Article", "title": "A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations", "authors": [ "Melissa Finlay", "Laurence A Hill", "Georgiana Neag", "Binal Patel", "Miruna Chipara", "Hannah C Lamont", "Kathryn Frost", "Kieran Patrick", "Jonathan W Lewis", "Thomas Nicholson", "James Edwards", "Simon W Jones", "Liam M Grover", "Amy J Naylor", "Melissa Finlay", "Laurence A Hill", "Georgiana Neag", "Binal Patel", "Miruna Chipara", "Hannah C Lamont", "Kathryn Frost", "Kieran Patrick", "Jonathan W Lewis", "Thomas Nicholson", "James Edwards", "Simon W Jones", "Liam M Grover" ], "abstract": "Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.", "keywords": [ "Osteocyte", "bone", "animal reduction", "3D model", "in vitro culture" ], "content": "\n\n\n\nScientific benefits\n\nAllows:\n\n• Study of osteoblast activity in long-term culture (>8 weeks).\n\n• Study of osteoblast to osteocyte differentiation.\n\n• Study of mineralisation and extracellular matrix production by osteoblasts.\n\n• Study of osteoblasts and osteocytes in a 3D, organotypic environment.\n\n3Rs benefits\n\n• Reduces the need for osteoblast and osteocyte isolation from vertebrates in skeletal research by supporting long-term culture of primary osteoblasts and secondary cell lines.\n\n• Provides a platform for osteocyte research in vitro, reducing the requirement for vertebrate in vivo experimentation.\n\nPractical benefits\n\n• Inexpensive, uses commonly available equipment and reagents.\n\n• Readily adaptable - can use cells of different origin and species.\n\n• Organotypic - 3D environment and matrix production and mineralisation performed by the osteoblasts.\n\nCurrent applications\n\n• Suitable for comparing phenotypes of osteoblasts isolated from genetically modified vertebrates or from patient samples.\n\n• Suitable for studying osteoblast and osteocyte function and for screening their response to therapeutic candidate compounds.\n\n• Suitable for long-term 3D culture of human osteoblast cell lines, such as hFOB, and for assessment and manipulation of their differentiation towards osteocytes.\n\nPotential applications\n\nOffers a platform for:\n\n• Studying osteoblast/osteocyte interactions with other cell types e.g. osteoclasts.\n\n• Screening of compound libraries to identify therapeutic candidate compounds.\n\n• Assessing efficacy of drugs and therapeutics under relevant conditions.\n\n\nIntroduction\n\nBone is a dynamic tissue that continually adapts throughout life to ensure maintenance of strength and integrity and to fulfil its many other functions, which include: adaptation to loading, preservation of mineral homeostasis (particularly calcium and phosphorous) and conservation of energy balance.1 These roles are made possible by the tightly controlled intercellular process called bone remodelling. Bone remodelling is a cyclical process that begins with tissue breakdown by bone resorbing osteoclasts, to release stored mineral and osteogenic factors such as bone morphogenic proteins and fibroblast growth factors.2 Resorption is followed by the formation of new bone tissue by osteoblasts. The balance between bone formation and bone resorption activity is critical for maintenance of tissue integrity. Likewise, this remodelling process allows bone to be repaired when damaged and to adapt rapidly to changes in physical demand, for example increased weight-bearing sports training or, oppositely, unloading through bed rest after injury and illness3 as well as from prolonged zero gravity experienced during space exploration.4\n\nThe precise coupling of osteoblast and osteoclast activity is tightly regulated by osteocytes (reviewed in Ref. 5). Up to 20% of osteoblasts differentiate into osteocytes6,7 which become entombed within newly deposited, immature bone tissue. Upon differentiation, they undergo a profound morphological change from a polygonal structure to a highly dendritic form. Entombed within individual lacunae, osteocyte dendrites extend through canaliculi to connect with other osteocytes, osteoblasts, osteoclasts, neurons, and the vasculature, creating a highly interconnected osteocyte-canalicular network. This distinguishing feature of osteocytes is a stipulation for their role as the main regulatory unit in bone metabolism and as orchestrators of bone cell activity.7,8\n\nDysregulation of bone remodelling is caused by multiple factors, the most notable of which is aging, which results in osteoporosis. As the global population ages, osteoporosis-induced fragility fractures are predicted to increase markedly – estimates made from six of the largest countries in Europe put the rise at 23.3% by 2030 compared to 2017.9 These figures give heightened urgency and a major push in research efforts to meet, or better yet curb, the impending demands for effective bone therapeutics.\n\nCurrently, bone researchers employ a range of platforms within in vivo, ex vivo and in vitro systems, all with their own advantages and disadvantages - detailed reviews of these platforms have been reported recently.10,11 In vivo bone models provide a complete and vascularised osseous system used to investigate bone metabolism, disease, healing, and development, as well as drug candidate efficacy and safety. However, these studies often require large sample numbers for moderate to severe procedures such as bone healing and ovariectomy-induced osteoporosis. Although ex vivo bone models, such as calvarial and femoral head culture, can be employed to investigate some of the same research questions as in vivo studies, the high running costs and intrinsic interspecies differences that exist when using animal tissue to study human disease limit their utility. Osteoblast and osteoclast in vitro cultures are widely employed, with multiple activity assessment assays and complex co-culture systems developed. However, they have been limited by the minimal success of osteocyte culture within these systems, and by the challenges of recapitulating the composition of inorganic mineral amalgamated to a complex hierarchy of collagen fibres that characterises native bone tissue in an in vitro system.12\n\nTo obtain a complete reflection of bone metabolic activity, the presence of interconnected osteocytes within any bone model is fundamental, however, isolation of primary osteocytes followed by cell culture under standard conditions is not successful and results in dedifferentiation to osteoblasts.13 Some immortalised osteocyte cell lines have been developed, which retain osteocyte morphology during 2D culture, for example the murine MLO-Y414 and the human HOB-01-C115 osteocytic cell lines. The challenges of in vitro osteocyte culture have led researchers to conduct a significant amount of bone research in vivo, with murine models being preferred due to lower running costs, simpler husbandry and genetic manipulability,16,17 as well as their rapid rate of bone turnover.18 In recent years, the movement towards replacing and reducing reliance on laboratory animals, and the requirement for in vitro systems for compound screening in drug development pipelines that are translatable, have renewed interest in the development of humanised in vitro bone assays capable of supporting osteocytes. Here we describe a three-dimensional, self-structuring bone model (SSBM) culture system that uses a biocompatible scaffold to support the differentiation of osteoblasts to osteocytes and the production of a complex collagen-rich mineralised matrix, that resembles native bone. We provide a detailed and descriptive protocol for preparing, setting up and maintaining SSBM constructs [Figure 1] and the analytical methods that have been developed specifically for their analysis.\n\nThe SSBM method is divided into 5 separate aspects (anchors, plates, hydrogel reagents, cell seeding and maintenance) each with their own steps for preparing, setting up and maintaining SSBM constructs. The minimum amount of preparation time required for setting up SSBM cultures is one week, providing all materials are available. However, many of the steps can be carried out up to six months in advance; this is recommended for stock preparation. Depending on the cell lines/types to be used for SSBM set up, preparing cells for cell seeding can vary and may take less than one week (i.e. faster proliferation rates). Cell seeding procedures on set up day will take 1-2 hours to complete. Maintenance steps within the first four weeks post SSBM set-up are performed on defined days after cell seeding but may vary depending on the cell type chosen. Analytical timepoints are selected at the researchers’ discretion, and dependent on the research question, with four weeks post SSBM set-up (i.e. just prior to introducing and maintaining osteogenic treatment) typically being the minimum analytical timepoint.\n\n\nMethods\n\nThis method (originally described in Ref. 19) relies on osteoblast-mediated contraction of a biocompatible scaffold (fibrin hydrogel) around a source of calcium and phosphate ions in the form of two cement anchors consisting of a combination of brushite (CaHPO4.2H2O) and B-TCP (Ca3(PO4)2). Contraction provides a 3D environment within which the osteoblasts, over time, replace the original fibrin matrix with organotypic collagen and facilitate its calcium phosphate mineralisation.\n\nThe original method19,20 utilised primary rat periosteal cells. The figures included here were generated using the human osteoblastic cell line, hFOB 1.19 (RRID: CVCL_3708) – selected based on their homogenous human origin, as well their stable karyotype,21 but the SSBM method can be readily adapted to accommodate osteoblasts derived from a number of different species, from primary osteoblasts isolated from vertebrate bone samples or from cell lines. We have used primary human osteoblasts and the human osteoblast cell line, hFOB 1.19,22 with similar success (data not shown). We note that primary cells have high levels of batch-batch variation in cell type populations and metabolic rates, as has been previously observed23 and that there are intrinsic differences in the proliferation rates of cell lines therefore, seeding densities and culture media composition should be adapted and validated on a case-by-case basis.\n\nThe following preparation, set up and maintenance methods have been separated into 5 distinct aspects, summarised in Figure 1.\n\n1. Anchor preparation\n\nNote – Sections 1.1-1.3 are prepared under non-sterile conditions\n\n1.1 Anchor mould preparation\n\nRequired:\n\n• Fused deposition modelling 3D Printer\n\n• Fusion 360 computer-aided design software (V.2.0) (Supplier: Autodesk, USA), or similar\n\n• 1.75 mm polylactic acid plastic filament (cat no.: 55318; supplier: Verbatim, Germany)\n\n• Perfection Plus Impress-E putty (Soft) (cat no.: 238-0084; supplier: Dental Sky Wholesaler Ltd., Kent (UK only))\n\nSteps:\n\n1. 3D print a positive frame of 1.75 mm polylactic acid plastic for the anchor moulds with the dimensions shown [Figure 2.A]. 3D Object (.3mf) and GCODE files are available as extended data.39\n\n2. Prepare the impression putty, as per the supplier’s instructions, and press the putty into the positive frame, ensuring the frame is completely filled.\n\n3. Allow the impress material to set for at least 24 hours at room temperature before removing from the mould. Make at least two. These are the anchor moulds for use in section 1.3.\n\n(A) A 3D sketch of a positive frame for the anchor mould, containing twenty equally spaced pegs, is drawn up with the dimensions provided above, using computer-aided design software, and a reusable frame is 3D printed with polylactic acid. (B) Dental impression material is pressed into the positive frame, ensuring the whole frame is filled, after which the mould is peeled out once completely set. (C) The recommended set up when preparing the β-TCP cement anchors. An ice tray is prepared with a spare anchor mould containing a single insect pin in each well placed in a top corner (black dotted box) and an empty mould in the adjacent bottom corner; as well as the orthophosphoric acid (3.5 M) in the vacant top corner. The β-TCP is weighed out and kept to one side of the weighing boat and placed in the vacant bottom corner of the ice tray (yellow dotted box) at a slight forward angle. The orthophosphoric acid is added to the same weighing boat as the β-TCP but kept separate prior to mixing. (D) When mixed, the resultant cement created, using the weight and volumes outlined [Section 1.3] is sufficient to fill 2-3 rows of the empty anchor mould, and pins from the spare anchor mould (C, black dotted box) are inserted immediately, prior to hardening (i). After repeating cement preparation, the cement is left to completely set within the anchor mould for approximately 3 days (ii).\n\n1.2 Orthophosphoric acid (3.5 M) preparation\n\nRequired:\n\n• Orthophosphoric acid (85-90% purity) (cat no.: 20624.262; supplier: VWR Chemicals, Leicestershire, UK)\n\n• Distilled water (d.H2O)\n\nSteps:\n\n1. Assuming 87.5% purity; for every 100 mL of Orthophosphoric acid:\n\nCRITICAL - Add the acid to water, never water to acid\n\n2. Store at 2-4 °C for up to 6 months.\n\n1.3 Calcium phosphate anchor preparation\n\nRequired:\n\n• Beta-tricalcium phosphate (β-TCP) (<125 μm particle size) (Plasma Biotal Limited., Tideswell, UK)\n\n• Orthophosphoric acid (3.5 M), prepared as described above [Section 1.2]\n\n• Austerlitz insect pins (stainless steel, 0.200 mm ø.) (cat no.: 26002-20; supplier: InterFocus Ltd., Linton, UK)\n\n• Anchor moulds (at least 2) [Section 1.1]\n\n• Fine metal tweezers\n\n• Metal Chattaway or flat-headed spatulas (~10 mm width)\n\n• Large weighing boats (100 mL) (cat no.: 611-9189; supplier: VWR)\n\n• Tray/bucket of ice\n\n• Paper towels\n\nNote - The recommended workspace set-up is shown [Figure 2.C.i] to ensure required equipment is within reach during anchor preparation.\n\nSteps:\n\n1. Using tweezers, add a single pin to each well of one of the spare anchor moulds [Figure 2.C.ii].\n\n2. Weigh out 1.25 ± 0.005 g of β-TCP into a large weighing boat (keep the powder to one side of the weighing boat [Figure 2.C.iii] then place the weighing boat on top of the ice tray.\n\n3. Add 500 μL orthophosphoric acid to the same weighing boat, but away from the powder [Figure 2.C.iii].\n\n• Tip - Point the weighing boat at a small angle down towards you.\n\n4. Mix the powder and acid together using the metal spatula, keeping the weighing boat on the ice, until the liquid mixture becomes a runny paste.\n\n• Note – there are batch-batch differences with β-TCP powder. Weigh out the mass stated above in [step 1] and adjust the volume of orthophosphoric acid by 100 μL increments/reductions until achieving the desired consistency (e.g. condensed milk).\n\n5. Quickly scrape and apply the paste onto 2-3 rows of the empty mould using the spatula, then submerge the spatula into the ice, followed by quickly transferring a single pin from the spare mould to each filled well. This must be completed before the cement hardens, which will take about 30 s [Figure 2.D.i].\n\n• Note – there will be an excess of cement on top of the mould during application, try to scrape off as much as possible, but a slight excess is permissible.\n\n• Tip – plunging the spatula into the ice helps with subsequent cleaning with a paper towel. If the excess cement is not readily removed with wiping, use hot soapy water.\n\n6. Clean the spatula (as above), then repeat steps 2-5 [Section 1.3] with the remaining wells in the mould, using a new weighing boat each time.\n\n7. Allow the anchors to set in the mould at room temperature for up to 3 days [Figure 2.D.ii], then remove from the mould and store in a sealed container at room temperature for up to 6 months.\n\n2. Plate preparation\n\nNote – Plates may be prepared under non-sterile conditions, as they are sterilised just prior to hydrogel preparation.\n\nRequired:\n\n• 6-well plate(s) (Cat no.: CLS3516; supplier: Corning, Flintshire, UK) – Tissue culture-treated plates are recommended, but not essential\n\n• Sylgard 184 silicone elastomer kit (Cat no.: 634165S; supplier: VWR)\n\n• Calcium phosphate anchors (12 per plate) prepared as described above [Section 1.3]\n\n• Large weighing boats\n\n• BD Emerald disposable syringe (10 mL) (Cat no.: 15205093; supplier: Fisher Scientific, Loughborough, UK)\n\n• BD PlastiPak disposable syringes (1.0 & 50 mL) (Cat nos.: 15489199 & 12651406; supplier: Fisher Scientific)\n\n• Tweezers\n\nSteps:\n\n1. Weigh out 10 ± 0.1 g of the base component of the elastomer kit into a weighing boat using a 50 mL syringe [Figure 3.A].\n\n2. Weigh out 1.0 ± 0.1 g of the curing component of the elastomer kit into the same weighing boat as the base component using a 1.0 mL syringe [Figure 3.A].\n\n3. Mix the base and curing components and slowly take up the mixture using a 10 mL syringe [Figure 3.A].\n\n• Tip – Use the end of the 10 mL syringe to mix the components.\n\n• Tip – Hold the weighing boat at an angle when taking up to allow the mixture to collect.\n\n• Tip – Continue mixing the components at regular intervals whilst taking it up.\n\n4. Add 1.5 mL of the mixture to each well of the plate [Figure 3.A].\n\n• Tip – If preparing more than one plate, weighing boats and syringes can be reused.\n\n5. Return the lids onto the plates and allow elastomer to set at room temperature for 7 days.\n\n• Note - Plates can be stored at room temperature after elastomer has set (no expiration has been found for using these plates).\n\n6. Once the elastomer has set, mark two anchor points on the underside of the plates, approximately 15 mm apart on each well [Figure 3.B.i].\n\n7. Bend the inserted pins towards the longer edge of the calcium phosphate anchor [Figure 3.B.ii] and insert into the elastomer at the marked point so that the bottom of the anchor sits on the top surface of the elastomer [Figure 3.B.iii].\n\n• Note – Plates with inserted anchors can be stored at room temperature and must be used within 6 months.\n\n(A) The elastomer base, followed by the curing agent, are weighed at a 10:1 ratio into the same weighing boat, and then thoroughly mixed with the end of an empty syringe. The mixture is then transferred to an empty 6-well culture plate using this same syringe and left to set for 7 days. (B) Once the elastomer has set, 2 β-TCP cement anchors are inserted into each well of the 6-well plate. Firstly, 2 marks are made 15 mm apart on the underside of the plate for each well (i). The pins within the β-TCP anchors are bent at a slight angle, towards the long edge of the trapezoidal base using tweezers (ii), then the pins are inserted at an angle into the elastomer (white arrows), using the measured points as guide, so that the base of the anchors sit on the top surface of the elastomer (white dotted line) (iii). (C) SSBM set up involves preparing the hydrogel by addition of the thrombin across each well of the plate, and ensuring the whole surface is covered by gently mixing and tapping the sides of the plate. Addition of fibrinogen straight into the centre of each well begins polymerisation and creation of the hydrogel. Cells are seeded directly on top of the polymerised hydrogel and cultured under normal culture conditions. (D) 3 days post-cell seeding, a scalpel is run around the walls of each well to detach any fibrin from the edge of the culture well. (i). When pipetting from the well, such as during media renewal, the plate is tilted forward to pool the liquid at the bottom to ensure that the fibrin matrix is not disturbed (ii). Created with Biorender.com.\n\n3. Reagent preparation for the fibrin hydrogel\n\n3.1 Phosphate buffered saline (PBS) solution\n\nRequired:\n\n• Gibco Phosphate buffered saline (PBS) tablets (1X) (cat no.: 18912014; supplier: Fisher Scientific,)\n\n• Distilled water (d.H2O)\n\nSteps:\n\n1. Dissolve PBS tablets in d.H2O at a ratio of 1 tablet per 500 mL d.H2O.\n\n2. Store at room temperature.\n\n3.2 Fibrinogen solution (20 mg/mL)\n\nRequired:\n\n• Fibrinogen (cat no.: F8630; supplier: Sigma-Aldrich, Gillingham, UK)\n\n• Ham’s F-12K (Kaighn’s) Medium (F-12K medium) (cat no.: 21127-022; supplier: Fisher Scientific)\n\n• BD Emerald disposable syringe (10 mL) (Cat no.: 15205093; supplier: Fisher Scientific)\n\n• 0.22-μm PES (Sterile) syringe filters (cat no.: E4780-1226; supplier: Starlab, Milton Keynes, UK)\n\n○ Note – Several PES filters might be required\n\nSteps:\n\n1. Place F-12K medium in 37 °C water bath for at least 20 mins.\n\n2. Weigh out at least 100 mg of fibrinogen (X) and use the following calculation to determine volume of media (Y) required:\n\n3. In a cell culture hood, dilute fibrinogen in calculated volume of F-12K medium - do not vortex.\n\n4. Place in a 37°C water bath for 1 to 2 hr, gently agitating every 30 min.\n\n5. In a cell culture hood, filter through 0.22-μm filters with the syringe, then store at -20 °C if not using straight away.\n\n3.3 Aprotinin (10 mg/mL)\n\nRequired:\n\n• Aprotinin (cat no.: A4529-10mg; supplier: Sigma-Aldrich)\n\n• PBS solution [Section 3.1]\n\n• BD Emerald disposable syringe (2.0 mL) (Cat no.: 15285083; supplier: Fisher Scientific)\n\n• 0.22-μm PES (sterile) syringe filters (cat no.: E4780-1226; supplier: Star Lab)\n\nSteps:\n\n1. Reconstitute entire contents of aprotinin vial using PBS solution at a ratio of 10 mg aprotinin to 1 mL PBS solution\n\n• Note – the above is for a 10 mg aprotinin vial. If purchased a different size, adjust resuspension volume accordingly to acquire 10 mg/mL\n\n• Tip – If acquired a 10 mg aprotinin vial, add 1 mL PBS solution directly into the aprotinin vial.\n\n2. Under sterile conditions, filter through 0.22-μm syringe filter with the syringe and prepare into 30 μL aliquots. Store at -20 °C.\n\n• Note - 1x 30 μL aliquots prepares 4x six-well plates. Avoid freeze-thaw cycles.\n\n3.4 Aminohexanoic acid (200 mM)\n\nRequired:\n\n• Aminohexanoic acid (cat no.: 07260-10g; supplier: Sigma-Aldrich)\n\n• PBS solution [Section 3.1]\n\n• BD Emerald disposable syringe (5.0 mL) (Cat no.: 15295083; supplier: Fisher Scientific)\n\n• 0.22-μm PES (sterile) syringe filters (cat no.: E4780-1226; supplier: Star Lab)\n\nSteps:\n\n1. Weigh out at least 50 mg aminohexanoic acid (X) and use the following calculation to determine volume of PBS required (Y):\n\n2. In a cell culture hood, resuspend in determined volume of PBS. Vortex until completely dissolved.\n\n3. In a cell culture hood, filter through 0.22-μm filter with the syringe and prepare into 30 μL aliquots. Store at -20 °C.\n\n• Note - 1x 30 μL aliquots prepares 4x six-well plates. Avoid freeze-thaw cycles.\n\n3.5 Thrombin (200 U/mL)\n\nRequired:\n\n• Thrombin, bovine (cat no.: 605157 – 1KU; supplier: Sigma-Aldrich)\n\n• Bovine serum albumin (BSA) (cat no.: A8806-5g; supplier: Sigma-Aldrich)\n\n• Ham’s F-12K (Kaighn’s) Medium (cat no.: 21127-022; supplier: Fisher Scientific)\n\n• 21G disposable needle (cat no.: SYR6232; supplier: SLS Scientific, Wilford, UK)\n\n• BD Emerald disposable syringe (2.0 mL) (Cat no.: 15285083; supplier: Fisher Scientific)\n\n• 0.22-μm PES (sterile) syringe filters (cat no.: E4780-1226; supplier: Star Lab)\n\n○ Note – The steps for thrombin preparation below is for a single 1 KU vial. Adjust accordingly if using a different quantity.\n\nSteps:\n\n1. Prepare 0.1% BSA solution by weighing out at least 6 mg BSA and use the following calculation to determine volume of F-12K medium required:\n\n2. Using the needle and syringe, pierce through the rubber stopper to reconstitute the entire content of the thrombin vial in 500 μL of 0.1% BSA solution\n\n3. Remove the syringe and invert the vial until the thrombin is completely dissolved, then remove the rubber stopper and reconstitute entire contents of vial in a single 4.5mL aliquot of 0.1% BSA solution.\n\n4. In a cell culture hood, filter the reconstituted thrombin through a 0.22-μm filter with the syringe and prepare 700 μL aliquots. Store the aliquots at -20 °C.\n\n• Note - A 700 μL aliquot is sufficient to prepare 4x six-well plates. Avoid freeze-thaw cycles.\n\n4. Preparing the fibrin hydrogel and construct set up\n\nRequired:\n\n• Thrombin (200 U/mL)\n\n• Aminohexanoic acid (200 mM)\n\n• Aprotinin (10 mg/mL)\n\n• Cell media (Non-osteogenic; refer to your cell supplier guidelines)\n\n• Fibrinogen solution (20 mg/mL)\n\n• Anchored plates\n\nSteps:\n\n1. Prior to cell seeding, spray the inside and outside of the plates with 70% ethanol and leave to dry in a sterile cell culture hood with the lids off\n\n2. Determine the number of 6-well plates to be prepared. See Table 1 to determine volumes required for thrombin solution preparation, based on the number of 6-well plates to be prepared.\n\n3. Thaw the appropriate number of aliquots of each of the above reagents and the fibrinogen solution and warm the media in a 37 °C water bath.\n\n4. Once thawed, in a cell culture hood, mix the thrombin solution reagents [Table 1] in their required quantities, based on the number of plates to be prepared and the volumes given in Table 1 below.\n\n5. Add 500 μL of thrombin solution across the elastomer surface between the two anchors of each well of the plate(s) - do not add onto the side of the wells. Ensure the culture surface of the well is completely covered [Figure 3.C].\n\n• Tip – Swirl and tap the sides of the plate to encourage even coverage, which can be challenging due to the hydrophobic nature of the elastomer.\n\n6. Add 200 μL of fibrinogen solution directly onto the thrombin solution between the two anchors of each well of the plate(s) - do not add onto the side of the wells. Immediately and gently, swirl the plate to ensure complete mixing of the fibrinogen and thrombin solutions [Figure 3.C].\n\n7. Incubate the plates at 37 °C in an incubator for 20-30 min to allow for polymerisation. If the intention is to use the plates immediately, during this polymerisation step, isolate the cells to be used and achieve the appropriate seeding density [Table 2].\n\n• Note - after polymerisation, plates can be stored at 2-8°C for up to a week. Preheat in an incubator at 37 °C for 30 mins prior to cell seeding.\n\n8. Seed 3.5 mL of cell suspension at the appropriate seeding density [Table 2] directly onto the fibrin hydrogel between the two anchors of each well of the plate(s) - do not add onto the side of the wells. Briefly swirl the plate and incubate under normal cell culture conditions.\n\nReagents and volumes required per 6-well plate to be prepared (Note - Volumes below account for an excess).\n\nSeeding density required for construct set up depends on the cell type being used. (Note - When using a new cell type, trial different seeding densities – in general, the faster the proliferation rate, the lower the seeding density required).\n\n** High variation between individuals.\n\n5. Maintenance of constructs\n\nRequired:\n\n• Disposable sterile scalpel (cat no.: 0511; supplier: Swann-Morton, Sheffield, UK)\n\n○ No specific blade size required – blade No. 24 used here\n\n• PBS solution [Section 3.1] – Autoclave sterilised\n\n• Trypsin-EDTA (2X) (cat no.: SLCC7608; supplier: Sigma-Aldrich)\n\n○ Note – Diluted from 10X stock conc. to 2X conc. using sterile PBS solution\n\n• Non-osteogenic cell culture media – refer to your cell supplier guidance\n\n• Osteogenic cell culture media – refer to your cell supplier guidance\n\nSteps:\n\n3-days post cell seeding – fibrin detachment\n\nThis step aids fibrin contraction by detaching any fibrin that may have adhered to the well walls\n\n1. Slowly run a scalpel around the edges of the wells at the top surface of the elastomer [Figure 3.D.i] to detach any fibrin that has not successfully retracted.\n\n• Tip – Run the scalpel from the 12 o’clock to the 6 o’clock points in the clockwise and anti-clockwise directions – do not run the scalpel around the whole well in one direction as this risks dislodging the fibrin from the anchors\n\n2. Remove 1.5 mL of media/well and replace with 1.5 mL of non-osteogenic cell culture media [Figure 3.D.ii]\n\n3. Continue to renew media every 3-4 days by removing and replacing 1.5 mL media/well. Ensure each well contains a minimum media volume of 3.5 mL\n\nAt 1 and 2 weeks post cell seeding – trypsin wash\n\nThis step is to prevent cell adherence and matrix formation outside of the fibrin construct\n\n1. Remove all media from each well and wash with 1.0 mL PBS. Discard the PBS.\n\n2. Add 0.5 mL of 2X Trypsin-EDTA to each well and incubate at 37 °C for 3 mins. Gently tap the sides of the plate and add 1.0 mL cell culture media.\n\n3. Remove entire volume of Trypsin-EDTA/media solution and replace with 3 mL non-osteogenic cell culture media.\n\n2-4 weeks post cell seeding – osteogenic incubation\n\nOsteogenic incubation can begin at the discretion of the user; commonly at 4 weeks post-cell seeding, or earlier if fibrin matrix width becomes < 3 mm\n\n1. Remove entire media volume from each cell culture well and replace with osteogenic media. Continue the normal media renewal regime using osteogenic media (refer to “3 days post-cell seeding – fibrin detachment”: Step 3).\n\n\nResults and analytical methods\n\nThe first significant observation is contraction of the fibrin matrix around the anchors [Figure 4.A.] which, depending on the cell source used, can occur in as little as 24 hours or up to 7 days post cell seeding. Cell-matrix interactions between the cell-bound integrins and Arg-Gly-Asp (RGD)-containing moieties, such as vitronectin, found within the extracellular matrix (ECM),24 are essential to cell behaviour, phenotype and survival. 2D culture platforms typically adhere cells through weak electrostatic forces on one side of the cell, which encourages an unnatural flattened conformation. By seeding cells in a 3D, naturally occurring matrix, the strong affinity between these integrins and ECM components on all surfaces of the cells results in complete enrobing of the cells in the matrix, which is observed as matrix contraction.25 Furthermore, with the presence of the two anchors in the well, the matrix contraction is controlled and forced into a cylindrical-like structure, whereas a disorganised spheroid will form in their absence.25 Because of the cylindrical matrix, the cells themselves are organised into a parallel and striated conformation [Figure 4B], which provides an in-built strain that further encourages cell differentiation and bone formation.\n\n(A) Once cells are seeded onto the fibrin hydrogel, contraction of the matrix around the two calcium phosphate anchors (green arrows) occurs. Upper and lower panels are the same images, with construct outline (dotted red line) excluded in the lower panel for clarity. Over time, the cells progressively form an organised and coordinated construct (as indicated by the narrowing of the red dotted rings). The resulting structure has a defined border-due to fibrin accumulation – which becomes stiffer and increasingly opaque following introduction of osteogenic conditions at 4 weeks. (B) Immunofluorescence staining of the cell nuclei (blue) and actin (red) shows that the cells present within the fibrin matrix align longitudinally between the two anchors, resulting in a striated configuration. This Z-stack maximum intensity projection image is of a construct cultured for 4-weeks. 20x magnification, scale bar is 100 μm. Stains: Hoechst (cat no.: 62249; supplier: Fisher Scientific) for nuclei (blue), phalloidin-488 (cat no.: A12379; supplier: Fisher Scientific) for actin (red). (C) Initially, mineral deposits are observed as dark specks (white dotted rings) of varying size within the matrix of the SSBM constructs (i). Following introduction of osteogenic media at 4 weeks, these become denser and more apparent (ii). Calcium phosphate anchor within red dotted ring. Images taken with light microscope; 4x magnification. Scale bars are 200 μm.\n\nOver time, the fibrin matrix contracts into a more defined structure, which then becomes increasingly opaque upon culturing under osteogenic conditions. This opacity is due to the occurrence of osteoid deposition by the cells and its subsequent mineralisation on the matrix [Figure 4.C].\n\nWith the correct planning, many analytical approaches can be carried out using a single construct, which demonstrates the analytical efficiency of the SSBM construct method; for example, Figure 5 shows the journey of a single construct through five separate analytical techniques. First the construct is stained intact using non-toxic ReadyProbes™ (cat no.: R37609; supplier: Invitrogen, Hertford, UK) for live-dead imaging. Subsequently, it is subjected to X-ray fluorescence (XRF) and then scanning electron microscopy (SEM). Meanwhile, the media collected from these constructs can be analysed by enzyme-linked immunosorbent assays (ELISA) at multiple timepoints throughout the construct culture period. Constructs stained with the ReadyProbes™ are also in an acceptable condition to be used for other analytical techniques, such as qPCR and immunofluorescence (IF) staining.\n\n(i) For analysis of exposed SSBM construct media, 3-4 days prior to the chosen analytical timepoint, intact SSBM constructs are isolated from their culture plates by cutting a strip of elastomer surrounding it, (ii) then incubated it in a fresh culture plate. At the timepoint, media can be collected and used for analysis, such as enzyme-linked immunosorbent assay (ELISA). The remaining construct can also be used for further analysis, such as live-dead imaging with ReadyProbes™ (cat no.: R37609; supplier: Invitrogen). Greater analytical efficiency can be achieved by using live-dead imaged constructs for other analytical approaches. To date, methods that have been optimised, and are under development, for SSBM construct analysis include quantitative polymerase chain reaction (qPCR), x-ray fluorescence (XRF), scanning electron microscopy (SEM) and immunofluorescent (IF) staining. Created with Biorender.com.\n\nSSBM isolation\n\nThe protocol above details the measures used to ensure cells attach to and remain within the fibrin matrix, and prevent cell adherence and proliferation occurring outside of the fibrin matrix – i.e. using hydrophobic Sylgard 184 silicone elastomer and regularly treating the wells with trypsin-EDTA. Although hydrophobic surfaces won’t completely prevent cell attachment (as previously demonstrated by the murine osteoblastic cell line, MC3T3), they can reduce cellular proliferation rate.26 Furthermore, trypsin-EDTA is a calcium chelator,27 which could affect the development of calcium-phosphate mineral, therefore it is used sparingly throughout the culture period. Given this, two separate cell populations exist within the culture dish - one outside and the other inside the SSBM construct. This is problematic for methods that involve analysing exposed media, (e.g. ELISA) as the results will reflect the activity of both cell populations, rather than just those within the SSBM construct. To overcome this problem, prior to media analysis, SSBM constructs must be isolated from their original culture plates and transferred into fresh plates. This can be achieved whilst retaining their shape integrity, and viability, by dissecting the elastomer as close as possible to the SSBM construct, with the anchors still attached, and placing it into a new 6-well dish [Figure 5]. Following culture in the appropriate media (i.e. complete or osteogenic media) for 3-4 days, the media is collected and used for analysis immediately, or stored for analysis at a later date (refer to manufacturer’s guidance for any specific preparation steps or storage requirements for cell supernatant, prior to beginning analysis or storing) – long-term storage at -80 °C is adequate for most analysis.\n\nLive-dead imaging\n\nThere are many approaches that can be used to determine cell viability based on their enzymatic and metabolic activity (e.g. MTT tetrazolium reduction, resazurin reduction and protease activity assays), or their membrane integrity (e.g. trypan blue exclusion and DNA binding dyes). However, these methods are often destructive, irreversible or cause long-term cytotoxicity. ReadyProbes™ penetrate the SSBM construct matrix and enable simple, as well as fast viability testing, without causing cell damage or cytotoxicity. Following manufacturer guidance, constructs still attached to their anchors and a strip of elastomer (as indicated above [Figure 5]) are resuspended and incubated for 30 min (33.5°C or 37 °C, 5% CO2) in 3.5 mL media with 2 drops/mL of NucBlue® Live and NucGreen® Dead reagents added directly, then imaged using a fluorescence microscope with standard DAPI (excitation/emission: 360/460 nm) and FITC/GFP filters (excitation/emission: 504/523 nm).\n\nRNA extraction for qPCR\n\nQuantitative polymerase chain reaction (qPCR) is a widely used technique that enables gene expression analysis in cells and tissue samples. During osteoblast differentiation, from progenitor cells through to osteocytogenesis, there are notable and patterned changes in the expression of specific genes, which have been reviewed extensively.28–30 Data from qPCR can be used as an indicator of osteoblast cell maturity within the SSBM construct, as well as to provide insights into how cells are responding to particular stimuli (e.g. bone formation modulators) and culture environment etc.\n\nAnalysis by qPCR (or RNA sequencing) relies on extraction of RNA of satisfactory quantity and quality, which is greatly influenced by the first steps in the extraction method; disruption and homogenisation of the cells and/or tissue lysates. This method has been developed and optimised for extracting good quality RNA from the SSBM constructs [Figure 6A], using the QIAGEN Mini RNEasy® Kit (cat no.: 74104; supplier: QIAGEN, Manchester, UK).\n\n(A) SSBM constructs are disrupted and homogenised prior to extraction of RNA for qPCR analysis, by following a process optimised for these constructs. Disruption involves vortexing samples, without the anchors, with glass beads, then by repeated aspiration using a syringe and needles. Disrupted samples are homogenised using QIAShredders (cat no.: 79656; supplier: QIAGEN), with the resultant sample ready for RNA extraction. (B) The elemental composition of the mineral present on SSBM constructs can be analysed using x-ray fluorescence (XRF). Whole constructs are first snap frozen in a dry ice bath using isopentane, immediately followed by dry freezing. XRF produces elemental map read-outs and energy spectra which can be used to locate and determine mineral distribution and maturity. The elemental maps shown here reveal co-localisation of calcium (red) and phosphorous (green), resulting in a yellow colouration, suggesting presence of a calcium phosphorous compound (yellow) i.e. bone-like mineral. Β-TCP anchors within white dotted outline. Scale bar is 2.0 mm. Created with Biorender.com.\n\nFirst, the SSBM construct must be unhooked from the anchors, then placed it in a 2.0 mL eppendorf tube containing 10-15 3 mm diameter glass beads (cat no.: Z143928-1EA; supplier: Sigma-Aldrich) and 600 μL of buffer RLT containing 10% β-mercaptoethanol (v/v) (cat no.: M6250; supplier: Sigma-Aldrich). Each sample is vortexed (bench top vortex) for 1-2 minutes, followed by shredding by repeated aspiration with a syringe first using a 16G (cat no.: 11532445; supplier: Fisher Scientific) needle, then a 19G needle (cat no.: 15341537; supplier: Fisher scientific), the construct and the buffer RLT mixture is repetitively aspirated with the syringe until the construct is a fine powder in suspension. Care is needed as a vacuum is created in the presence of large pieces of construct. To reduce RNA loss, the same syringe is used between needles for the same sample; but to prevent cross contamination, a new syringe and set of needles are used for each individual SSBM construct. The resultant lysate is transferred to a QIAshredder spin column (cat no.: 79654; supplier: QIAGEN) and centrifuged (2 min; max speed in a microcentrifuge), followed by a repeat centrifugation of the resultant supernatant, with the shredder removed and collection tube capped. The supernatant is transferred by careful pipetting - to ensure no pelleted debris is taken up - to a new 1.5 mL Eppendorf and then used for RNA extraction following the steps outlined by the QIAGEN RNEasy® Mini handbook, beginning with addition of 70% ethanol to the supernatant (section “Protocol: Purification of Total RNA from Animal Tissues”, pages 51-52, as of October 2019). Typical RNA yields for hFOB 1.19 are shown in Table 3, with the purity of the RNA extracted deemed acceptable when the ratio of absorbance at 260 and 280 nm is 2.0 ± 0.15.\n\nNote - resultant yields will vary considerably with the culture period time and culture conditions, the cell type used to create the SSBM constructs and user experience. Values are the range obtained from successful RNA extractions performed previously (sample number, n, given for each timepoint).\n\nFixation\n\nThe structure of SSBM constructs must be preserved at the elected timepoints to obtain an accurate snapshot of structural development and changes. Like bone, SSBM constructs are composite “tissues”, containing an organic and an inorganic phase, which require different methods of preservation. SSBM constructs that are to be used for structural analysis (for either the organic or inorganic phase) must first be fixed, whilst remaining attached to the anchors (as indicated above [Figure 5]). This begins with an incubation of 4.0 mL/construct 4% formaldehyde (cat no.: HT501128; supplier: Sigma-Aldrich) for 4 hours on a shaker plate at 4.0 °C; followed by three washes with 4.0 mL/construct PBS. Constructs can then be stored in PBS at 4.0 °C for up to 2 weeks. Fixation of SSBM constructs by paraformaldehyde preserves the organic phase whilst retaining the inorganic mineral phase.\n\nFreeze-drying\n\nFor structural analysis that particularly concerns the inorganic phase of the SSBM constructs, moisture must be removed from samples. Freeze-drying (lyophilisation) is the preferred method to remove water from SSBM constructs, as it maintains sample structure (both microscopically and chemically) better than alternative approaches such as oven-drying.31,32\n\nSSBM constructs must first be frozen rapidly, as slow freezing can cause crystallisation of the ice, which will distort and damage the samples. This can be achieved using liquid nitrogen immersion, however, with the SSBM constructs, this can be too vigorous and can result in damage to the fragile constructs. A more successful alternative is to freeze in an isopentane bath [Figure 6.B]. Place a glass beaker, half-filled with isopentane and a small amount of dry ice, into a container of dry ice, ensuring the beaker is surrounded completely. SSBM constructs attached to their anchors and a strip of elastomer (as indicated above [Figure 5]) are placed in an Eppendorf tube and submerged in the isopentane beaker for 5 minutes. Ensure that the Eppendorf is completely submerged and add additional dry ice pellets to the isopentane if the bubbling begins to slow/stop. The submersion time can be increased if the construct is not completely frozen after 5 minutes. Once frozen, constructs are stored at -80 °C, or immediately freeze-dried (Lablyo mini, York, UK) at -50 °C for at least 4 hours. Freeze-dried constructs can be stored in a sealed container at room temperature, away from moisture, for up to 6 months.\n\nX-ray fluorescence\n\nX-ray fluorescence (XRF) is an inexpensive, non-destructive and rapid analytical approach to investigate the elemental composition of samples.33–36 In this case, the location and extent of calcium and phosphorous within the SSBM constructs can be assessed. Constructs are placed in an XRF (M4 Tornado, Bruker Nano Gmbh, Germany) under a 20 mBar vacuum. After adjusting the focus on the construct, the region of interest (ROI) is outlined and then scanned with the X-ray (50 kV, 400 uA) at 1000 spots/10 mm (spot size = 25 μm), 5 ms/pixel exposure time.\n\nBy overlaying the calcium and phosphorous elemental maps [Figure 6.B], the resultant yellow colouration indicates co-localisation of the two elements, which strongly suggests the presence of calcium-phosphate mineral compounds. The resultant maps and spectra provide further information regarding the distribution and maturity of the mineral present through the varying degrees of colour intensity visualised on the maps, which are determined by the size of each element’s unique energy signal. Regarding the SSBM constructs, mineral distribution and maturation appears heterogeneous as found in in vivo bone tissue.37\n\nImmunofluorescence (IF) staining techniques offer the capability to visualise the structural and biological characteristics of samples with high resolution and accuracy. The ability to section samples [Figure 7.A.i] and the vast range of fluorescent secondary antibodies available means a single sample can be analysed for numerous structural and biological markers of interest, thus furthering analytical efficiency. Furthermore, SSBM constructs can also be IF stained intact, enabling a gross overview of the cellular orientation, as well as the expression of markers of interest in relation to their location within the construct within the construct [Figure 4.B, Figure 7.A.ii].\n\nFollowing adapted protocols for an established gelatin-based embedding, sectioning, and immunofluorescent staining protocol37 (i) and one general protocol optimised for sectioned collagen-based matrices (ii), respectively, resultant 50 μm sections (i) and whole intact (ii) SSBM constructs are used to examine specific bone cell markers e.g., podoplanin (green), within the SSBM constructs, as well as an overview of cell presence and conformation. Left to right: Images above are z-stack maximum intensity projections of cultured SSBM constructs, using phalloidin for actin staining (red) and hoechst nuclei staining (blue). Scale bars are 50 μm.\n\nPrior to staining, constructs must be fixed and then unhooked from their anchors – the constructs tend to retain their shape after unhooking once they have been fixed, as well as with increasing maturity (i.e. older constructs are stiffer).\n\nIF Staining: Sectioned constructs\n\nThe method developed by Kusumbe et al., 201538 reliably allows the preparation of bone tissue for high-resolution IF images without compromising tissue structure or proteins. Due to the delicate and bone-like nature of the SSBM constructs, this method is also ideal for their IF staining.\n\nThe method begins with sample fixation steps, which have been adapted and described above for the SSBM constructs (refer to 'structural analysis' above), followed by decalcification and cryoprotection steps in preparation for embedding. Unlike traditional methods of embedding (e.g. paraffin embedding), this method uses gelatin-based embedding, with polyvinylpyrrolidone (PVP) and sucrose cryoprotection, as it has been found to prevent bone disintegration and allows for thicker sectioning. However, due to the fragility of the SSBM constructs in comparison to bone (i.e. a thinner composite tissue containing collagen and fibrin), the incubation conditions required for embedding are altered from 45 mins at 60 °C, to 90 mins at 45 °C. Once embedded and frozen, the protocol38 outlines the steps for sectioning the resultant tissue blocks. In the case of the SSBM constructs, 50 μm sections are recommended [Figure 7.A.i]. Finally, the method outlines the IF staining procedure. Antibody incubation durations, dilutions, and fluorophore panels are subject to the researcher’s discretion. Figure 7.A.i shows SSBM constructs stained for podoplanin using an anti-human podoplanin rabbit monoclonal antibody (1:200) (cat no.: D9D7; supplier: Cell Signaling Technology, USA), followed by an Alexa Fluor® 647 AffiniPure donkey anti-rabbit IgG secondary (1:200) (cat no.: 711-605-152; supplier: Jackson ImmunoResearch, Ely, UK). Along with hoechst (1:400) (cat no.: 62249; supplier: Fisher Scientific) for nuclei and phalloidin-488 (1:100) (cat no.: A12379; supplier: Fisher Scientific) for actin. Once stained, the SSBM construct sections are treated with ProLong™ Diamond Antifade Mountant (cat no.: P36961; supplier: Fisher Scientific) then coverslipped.\n\nIF Staining: Whole constructs\n\nA general IF staining protocol developed for collagen-based matrices, that have been paraffin-embedded and sectioned, has been developed in our laboratories and forms the basis of the method that is used for staining SSBM constructs whole and intact [Figure 7.A.ii].\n\nSSBM constructs that have been unhooked from their anchors, are decalcified and cryoprotected as described by Kusumbe et al., 2015.38 In a fresh 6-well plate, SSBM constructs are treated with blocking buffer [Table 4] for 1 hour at room temperature (RT) – ensuring the constructs are completely submerged (>3.0 mL solution per construct) – then washed twice with PBS. Primary antibody is diluted accordingly using antibody buffer [Table 4] and >3.0 mL solution per construct added to each well, then incubated for 2 hours at RT. Constructs are washed 3 times with PBS and treated with the secondary antibody, as well as hoechst (1:400) and phalloidin (1:100), diluted accordingly in antibody buffer [Table 4], at >3.0 mL solution per construct for 2 hours at RT and covered. Finally, the SSBM constructs are washed 3 times with PBS and are immediately imaged on a confocal microscope, not in solution, on a glass-bottomed (170±5 μm thickness) imaging dish (cat no.: 80137; supplier: Ibidi, Germany).\n\n\nConclusion\n\nThis detailed step-by-step protocol for the preparation, production, and maintenance of a novel 3D in vitro bone model, and the adapted analytical methods described, can accommodate various osteoblastic cell sources and species. Importantly, we have now demonstrated the utility of this model using hFOB 1.19 cells (as shown here), and previously with primary rodent osteoblasts,19,20 as well as with primary human osteoblasts (data not shown). This inherent flexibility in cell source, and the minimal requirement for specialist equipment/reagents (beyond those available in most well-resourced molecular biology laboratories) widens the potential applications and accessibility for this model. The use of cell lines or, where available, primary human cells rather than primary cells from other vertebrate species, should be used to reduce the requirement for animals as the osteoblast source.\n\nThe humanisation of the method using the hFOB cell line has reduced our requirement for ex vivo samples considerably. We estimate that the work conducted over the past two years in our laboratory (during which time, a total of >280 hFOB-containing constructs have been produced) would have required tissue from >140 rats using the previous ex vivo iteration of the method.19 Within the UK, there are at least 6 research groups working on bone modelling currently using animal-derived primary tissue models. Considering a similar level of animal use to our laboratory, we estimate that the development of our humanised in vitro model for the long-term study of bone has the potential to replace over 840 rodents per year for similar research across the UK alone. With the development potential and adaptability of the model to study normal development, as well as bone diseases and novel therapies, the replacement potential for this in vitro model is significant.\n\nWe anticipate that this assay could be readily adapted to incorporate mixed cell populations to study cell-cell interactions. For example, co-culture with osteoclasts to study bone remodelling or with malignant cells to study bone metastasis. Assessment of the effects of candidate therapeutic compounds on construct mineralisation rate and osteocyte differentiation and function will allow screening for efficacy prior to in vivo studies. Such uptake of this method at the discovery stage of research, particularly with the capacity to utilise human and patient cells, will lead to a reduced requirement for in vivo and ex vivo platforms, therefore further replacing the use of laboratory animals, and improve the translatability of preclinical studies.", "appendix": "Data availability\n\nZenodo: A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations. https://doi.org/10.5281/zenodo.7620806 39\n\nThis project contains the RNA yields data and original image files.\n\nZenodo: A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations. https://doi.org/10.5281/zenodo.7620806 39\n\nThis project contains the following extended data:\n\n- 6-well 20 x anchors Mold Frame.gcode\n\n- 6-well 20 x anchors.3mf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to give their appreciation and gratitude to the NC3Rs for funding and supporting the research needed for the development of this protocol. Dr Triin Major (Birmingham Tissue Analytics, University of Birmingham) and Miss Julia Manning (Institute of inflammation and Ageing, University of Birmingham) for immunofluorescence staining and imaging support. Dr Erik Hughes and Mr Adam McGuinness (School of Chemical Engineering, University of Birmingham) for x-ray fluorescence support.\n\n\nReferences\n\nConfavreux CB: Bone: from a reservoir of minerals to a regulator of energy metabolism. Kidney Int. Suppl. 2011 Apr [cited 2022 Nov 9]; 79(S121): S14–S19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRucci N: Molecular Biology of Bone Biology. Clin. Cases Miner. Bone Metab. 2008; 5(1): 49–56. PubMed Abstract\n\nRittweger J, Frost HM, Schiessl H, et al.: Muscle atrophy and bone loss after 90 days’ bed rest and the effects of flywheel resistive exercise and pamidronate: results from the LTBR study. Bone. 2005 Jun [cited 2022 Nov 21]; 36(6): 1019–1029. Publisher Full Text Reference Source\n\nStavnichuk M, Mikolajewicz N, Corlett T, et al.: A systematic review and meta-analysis of bone loss in space travelers. npj Microgravity. 2020 May 5 [cited 2023 Jan 13]; 6(1): 1–9. Publisher Full Text Reference Source\n\nBonewald LF: The amazing osteocyte. J. Bone Miner. Res. 2011 Feb 1 [cited 2020 Dec 8]; 26(2): 229–238. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranz-Odendaal TA, Hall BK, Witten PE: Buried alive: How osteoblasts become osteocytes. Dev. Dyn. 2006 Jan 1 [cited 2020 Mar 29]; 235(1): 176–190. PubMed Abstract \| Publisher Full Text\n\nBellido T: Osteocyte-driven bone remodeling. Calcif. Tissue Int. 2014; 94(1): 25–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonewald LF: Generation and function of osteocyte dendritic processes. J. Musculoskelet. Neuronal Interact. 2005; 5(4): 321–324.\n\nBorgström F, Karlsson L, Ortsäter G, et al.: Fragility fractures in Europe: burden, management and opportunities. Arch. Osteoporos. 2020 Dec 1 [cited 2020 Nov 5]; 15(1): 59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhnert S, Rinderknecht H, Aspera-Werz RH, et al.: Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models. Arch. Toxicol. 2020 Dec 1 [cited 2021 Nov 30]; 94(12): 3937–3958. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellido T, Delgado-Calle J: Ex Vivo Organ Cultures as Models to Study Bone Biology. JBMR Plus. 2020 Mar 1 [cited 2022 Nov 21]; 4(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark Y, Cheong E, Kwak JG, et al.: Trabecular bone organoid model for studying the regulation of localized bone remodeling. Sci. Adv. 2021 Jan 20 [cited 2022 Nov 21]; 7(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernhardt A, Österreich V, Gelinsky M: Three-Dimensional Co-culture of Primary Human Osteocytes and Mature Human Osteoclasts in Collagen Gels. Tissue Eng. - Part A. 2020 Jun 1 [cited 2021 Nov 30]; 26(11–12): 647–655. PubMed Abstract \| Publisher Full Text\n\nPapanicolaou SE, Phipps RJ, Fyhrie DP, et al.: Modulation of sclerostin expression by mechanical loading and bone morphogenetic proteins in osteogenic cells. Biorheology. 2009 [cited 2020 Mar 30]; 46(5): 389–99. PubMed Abstract \| Publisher Full Text\n\nBodine PVN, Vernon SK, Komm BS: Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone. Endocrinology. 1996 Nov 1 [cited 2023 Jan 13]; 137(11): 4592–4604. PubMed Abstract \| Publisher Full Text Reference Source\n\nLelovas PP, Xanthos TT, Thorma SE, et al.: The Laboratory Rat as an Animal Model for Osteoporosis Research. Comp. Med. 2008 Oct [cited 2022 Nov 21]; 58(5): 424. Free Full Text\n\nTurner AS: Animal models of osteoporosis--necessity and limitations. Eur. Cell. Mater. 2001 [cited 2022 Nov 21]; 1: 66–81. PubMed Abstract \| Publisher Full Text\n\nTakakura A, Takao-Kawabata R, Isogai Y, et al.: Differences in vertebral, tibial, and iliac cancellous bone metabolism in ovariectomized rats. J. Bone Miner. Metab. 2016 May 1 [cited 2021 Dec 5]; 34(3): 291–302. PubMed Abstract \| Publisher Full Text\n\nIordachescu A, Williams RL, Hulley PA, et al.: Organotypic Culture of Bone-Like Structures Using Composite Ceramic-Fibrin Scaffolds. Curr. Protoc. Stem Cell Biol. 2019 Feb 1 [cited 2020 Nov 12]; 48(1): e79. PubMed Abstract \| Publisher Full Text\n\nIordachescu A, Amin HD, Rankin SM, et al.: An in vitro Model for the Development of Mature Bone Containing an Osteocyte Network. Adv. Biosyst. 2018; 2(2). Publisher Full Text\n\nSubramaniam M, Jalal SM, Rickard DJ, et al.: Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ERα cells: Bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization. J. Cell. Biochem. 2002 [cited 2020 Mar 6]; 87(1): 9–15. PubMed Abstract \| Publisher Full Text\n\nHarris SA, Enger RJ, Riggs LB, et al.: Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J. Bone Miner. Res. 1995 Feb [cited 2020 Mar 6]; 10(2): 178–186. PubMed Abstract \| Publisher Full Text\n\nKaur G, Dufour JM: Cell lines: Valuable tools or useless artifacts. Spermatogenesis. 2012 Jan [cited 2022 Nov 21]; 2(1): 1–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Flier A , Sonnenberg A: Function and interactions of integrins. Cell Tissue Res. 2001 [cited 2022 Nov 21]; 305(3): 285–298. Publisher Full Text\n\nWudebwe UNG, Bannerman A, Goldberg-Oppenheimer P, et al.: Exploiting cell-mediated contraction and adhesion to structure tissues in vitro. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2015; 370: 20140200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLourenço BN, Marchioli G, Song W, et al.: Wettability influences cell behavior on superhydrophobic surfaces with different topographies. Biointerphases. 2012 Jul 26 [cited 2022 Nov 21]; 7(1–4): 1–11. PubMed Abstract \| Publisher Full Text\n\nLai TY, Cao J, Ou-Yang P, et al.: Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand. Sci. Reports. 2022 Apr 5 [cited 2022 Nov 21]; 12(1): 1–8. Publisher Full Text Reference Source\n\nChen X, Wang L, Zhao K, et al.: Osteocytogenesis: Roles of Physicochemical Factors, Collagen Cleavage, and Exogenous Molecules. Tissue Eng. Part B Rev. 2018 Jun 6 [cited 2022 Nov 21]; 24(3): 215–225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonewald LF: Osteocytes.Marcus R, Feldman D, Nelson D, et al., editors. Osteoporosis. 3rd ed.Academic Press; 2008; pp. 169–89.\n\nDallas SL, Bonewald LF: Dynamics of the Transition from Osteoblast to Osteocyte. Ann. N. Y. Acad. Sci. 2010 [cited 2022 Nov 21]; 1192: 437–443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÁlvarez-Castillo E, Bengoechea C, Felix M, et al.: Freeze-Drying versus Heat-Drying: Effect on Protein-Based Superabsorbent Material. Process. 2021 Jun 21 [cited 2022 Nov 21]; 9(6): 1076. Publisher Full Text Reference Source\n\nElavarasan K, Shamasundar BA: Effect of oven drying and freeze drying on the antioxidant and functional properties of protein hydrolysates derived from freshwater fish (Cirrhinus mrigala) using papain enzyme. J. Food Sci. Technol. 2016 Feb 1 [cited 2022 Nov 21]; 53(2): 1303–1311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanssens K, Vittiglio G, Deraedt I, et al.: Use of Microscopic XRF for Non-destructive Analysis in Art and Archaeometry. X-Ray Spectrom. 2000; 29(1): 73–91. Publisher Full Text\n\nAnitha A, Brasoveanu A, Duarte M, et al.: Restoration of X-ray fluorescence images of hidden paintings. Signal Process. 2013 Mar 1; 93(3): 592–604. Publisher Full Text\n\nStockmann U, Cattle SR, Minasny B, et al.: Utilizing portable X-ray fluorescence spectrometry for in-field investigation of pedogenesis. Catena. 2016 Apr 1; 139: 220–231. Publisher Full Text\n\nSpecht AJ, Lin Y, Weisskopf M, et al.: XRF-measured bone lead (Pb) as a biomarker for Pb exposure and toxicity among children diagnosed with Pb poisoning. Biomarkers. 2016 May 18 [cited 2022 Nov 21]; 21(4): 347–352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalbach-Hirsch J, Rauner M, Hofbauer C, et al.: New insights into the role of glycosaminoglycans in the endosteal bone microenvironment. Biol. Chem. 2021 Oct 1 [cited 2022 Nov 21]; 402(11): 1415–1425. PubMed Abstract \| Publisher Full Text\n\nKusumbe AP, Ramasamy SK, Starsichova A, et al.: Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue. Nat. Protoc. 2015 Oct 29 [cited 2021 Dec 5]; 10(12): 1904–1914. PubMed Abstract \| Publisher Full Text Reference Source\n\nFinlay M, Hill L, Neag G, et al.: A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations. Zenodo. 2023. Publisher Full Text" }	[ { "id": "189266", "date": "19 Feb 2024", "name": "Jesus Delgado-Calle", "expertise": [ "Reviewer Expertise osteocyte", "bone biology", "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\nIn this manuscript, Finlay and colleagues provide a detailed, step-by-step protocol to establish 3D cultures of osteoblastic cells. The rationale for developing a 3D in vitro system for long culture of osteoblasts and their differentiation to osteocytes is strong. The description of the procedures is thorough and comprehensive. Specific comments to the authors are shown below:\nThe authors indicate this is an inexpensive method doable for most labs. However, the first steps of the process require the use of a 3D printer. The authors need to tone down their statements about how easy and inexpensive this method is. Is there an alternative to the 3D printed mould?\n\nHOB-01-C1 is not an osteocytic cell line, is a pre-osteocyte-like cell line.\n\nThe manuscript would benefit from the inclusion of a deeper discussion of the different 3D models and ex vivo models currently available to study osteocyte biology and a description of the major advantage(s) of using the described system.\n\nThe authors suggest this model can substitute in vivo approaches. This statement needs to be toned down, as the system as presented does not represent the complexity of the bone microenvironment. Even a co-culture between osteoblasts and osteoclasts/tumor cells will only represent a minor fraction of the cells involved in bone biology.\n\nThe reader would benefit from the inclusion of clear visual examples of the morphology of osteocytes generated in this in vitro system.\n\nThe osteogenic step is not clearly defined. Reagents and concentrations, as well as treatment time, need to be indicated.\n\nThe protocol seems to take a long time, increasing user error and the chance of contamination. Advice on how to avoid contamination needs to be provided.\n\nAre the 3Rs implications of the work described accurately? Partly\n\nAre a suitable application and appropriate end-users identified? 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": [ { "c_id": "11488", "date": "21 Jun 2024", "name": "Amy Naylor", "role": "Author Response", "response": "The authors indicate this is an inexpensive method doable for most labs. However, the first steps of the process require the use of a 3D printer. The authors need to tone down their statements about how easy and inexpensive this method is. Is there an alternative to the 3D printed mould? Author Response: Whilst 3D printing is not available to everyone, it does not require expensive equipment or maintenance in comparison to typical lab equipment and reagents and once trained, is easy to use. Alternatively, there are multiple companies who will print the frame at very low cost e.g., £3 per frame as of March 2024. Furthermore, once the frame is printed, it can be reused multiple times to create many anchor moulds. To support the reader in navigating this, we have added the following text to the relevant methods section: “Note - if unable to 3D print on site, there are several professional online printing services who can print the frame quickly and cost effectively from the 3D object file. Recommendation to print 1-3 frames.\" \"Note - once printed, positive frames can be reused multiple times (until anchor shape has become distorted). Shelf life: >4 years.\" HOB-01-C1 is not an osteocytic cell line, is a pre-osteocyte-like cell line. Author Response: Agreed, we have changed the wording of the sentence to more correctly reflect this. The manuscript would benefit from the inclusion of a deeper discussion of the different 3D models and ex vivo models currently available to study osteocyte biology and a description of the major advantage(s) of using the described system. Author Response: We agree and have added a section to introduction that expands upon this point and have referenced recent reviews that discuss this in depth. The authors suggest this model can substitute in vivo approaches. This statement needs to be toned down, as the system as presented does not represent the complexity of the bone microenvironment. Even a co-culture between osteoblasts and osteoclasts/tumor cells will only represent a minor fraction of the cells involved in bone biology. Author Response: We agree with the reviewer, however it is not our ambition with this model to recapitulate the complexity of the bone microenvironment, rather to create a long-term in vitro environment that can successfully support osteoblasts. We have acknowledged in the conclusion that this culture cannot replicate the bone microenvironment in its entirety but that it adds to the in vitro options available to researchers in their endeavours to reduce reliance on in vivo models, particularly when it comes to the toxicity and pharmacokinetic testing phases. We have also highlighted the properties that can allow our model to be made into more complex in vitro models and we encourage the reader to use this method to do further research into this. We have also highlighted the unique feature of our model in the introduction, namely the in-built strain achieved via the anchors, which we believe is crucial to its recapitulating the bone matrix microenvironment. The reader would benefit from the inclusion of clear visual examples of the morphology of osteocytes generated in this in vitro system. Author Response: We recognise the reason for the reviewer’s comment. In this manuscript we make the case that this model is capable of long-term culture of osteoblasts and their differentiation towards osteocytes. In response to the reviewer’s comment, we have amended the language within the manuscript to clarify that this method supports the differentiation of osteoblasts towards osteocytes, but we stop short of demonstrating evidence of mature osteocyte formation. Of note, images of osteocytes within the constructs have been previously published in Iordachescu et al. 2018, albeit with a different cell source, which is referenced frequently throughout the manuscript. Given that the aim of this manuscript was to provide a detailed and practical methodology for a previously published method, we consider that these previously published images are sufficient. Of note, we are currently preparing another manuscript that will characterise the cellular biology of hFOB1.19 in SSBM over time in more detail. The osteogenic step is not clearly defined. Reagents and concentrations, as well as treatment time, need to be indicated. Author Response: We agree. Thank you for recommending this clarification. We have edited the table with suggested cell line seeding densities to include suggested osteogenic media, whilst making clear that this is at the discretion of the user, and we have added additional information in section 5 of the methodology. The protocol seems to take a long time, increasing user error and the chance of contamination. Advice on how to avoid contamination needs to be provided. Author Response: Unfortunately, this protocol does indeed take time. However, in our experience there is no greater risk of cell culture contamination than with the maintenance of e.g., cell lines, provided stringent aseptic technique and conditions are adhered to. We have included a list of simple suggestions to reinforce this message, such as frequent decontamination of incubator and culture hood, separate pipettes, regular media filtration etc." } ] }, { "id": "189268", "date": "21 Mar 2024", "name": "Holger Jahr", "expertise": [ "Reviewer Expertise 3D models", "bone regeneration", "molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present paper describes an in vitro culture method for the long-term analysis of 3D bone models from osteoblastic cells or cell lines. The manuscript is logically structured and well written. Despite its complexity, it leaves me with rather few questions or suggestions for improvement, respectively.\n\nPer convention, extracted RNA is used in RT-qPCR, not qPCR. This should be corrected. Was the 260/230 nm ratio determined, too?\n\nDuring plate preparation (#2), each well is pre-filled with 1.5 mL Sylgard 184 silicone elastomer solution. Subsequently, the fibrin hydrogel is prepared (#4) by adding 0.2 mL of fibrinogen solution (20 mg/mL) to 0.5 mL of thrombin solution “across” the elastomer surface, is the final concentration 8 mg/mL? Does the mixture diffuse into the elastomer to potentially alter the final concentration?\n\nLater, 3.5 mL of cell suspension (about 525,000 cells) are seeded onto the fibrin hydrogel. Did you try using RNeasy Micro kit, too?\n\nI am not convinced that Fig. 7 suggests the presence of osteocytes in the tissue through a single podoplanin-positive staining in the absence of controls as this protein is also expressed on other cell types. IF staining should include other accepted bone-specific or bone maturation makers. This could be easily checked and should be considered.\n\nImportantly, while using XRF is a nice idea, demonstrating calcium and phosphorous within the SSBM constructs does not make this a “bone model”. To this end, a major limitation of the present methodology is that no biomineralization has been shown, yet, while the title suggests a “bone model” was developed. Stating that “mineral distribution and maturation appears heterogeneous as found in in vivo bone tissue” does not really convince me. Please provide further evidence.\n\nAre the 3Rs implications of the work described accurately? No\n\nAre a suitable application and appropriate end-users identified? 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": [ { "c_id": "11489", "date": "21 Jun 2024", "name": "Amy Naylor", "role": "Author Response", "response": "Per convention, extracted RNA is used in RT-qPCR, not qPCR. This should be corrected. Was the 260/230 nm ratio determined, too? Author Response: Thank you for this comment. We confirm that we did indeed perform reverse transcription PCR, therefore we have amended the text to RT-qPCR, as suggested. 260/230 ratios were not collected because no Trizol or phenol were used during sample isolation. The 260/280 ratio was collected to give an indication of RNA quality and to detect other potential contaminants. In all cases we found this ratio to be close to 2 (a tolerance of +0.15 was considered acceptable), demonstrating reasonable purity. During plate preparation (#2), each well is pre-filled with 1.5 mL Sylgard 184 silicone elastomer solution. Subsequently, the fibrin hydrogel is prepared (#4) by adding 0.2 mL of fibrinogen solution (20 mg/mL) to 0.5 mL of thrombin solution “across” the elastomer surface, is the final concentration 8 mg/mL? Does the mixture diffuse into the elastomer to potentially alter the final concentration? Author Response: There is a period between addition of elastomer and addition of fibrin mixture where the elastomer is left to set and solidify, therefore diffusion is not expected to occur between the fibrin matrix and the elastomer base. This is described in section 2, “plate preparation”, step 5 as follows: “Return the lids onto the plates and allow elastomer to set at room temperature for 7 days.” We have now added the word “solid” to make this clearer for the reader such that the sentence now reads: “Return the lids onto the plates and allow elastomer to set solid at room temperature for 7 days.” Later, 3.5 mL of cell suspension (about 525,000 cells) are seeded onto the fibrin hydrogel. Did you try using RNeasy Micro kit, too? Author Response: The average weight of the constructs is approximately 50 mg, however, because they are formed from a hydrogel, this weight can vary dependent on the degree of water absorbed by/contained within it. The Qiagen product information ( https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-plus-kits ) states that “the RNeasy Plus Micro Kit purifies total RNA from up to 5 x 105 cells or 5 mg tissue, and the RNeasy Plus Mini Kit isolates total RNA from up to 107 cells or 30 mg tissue.” Given this, we chose to use the mini rather than micro kit, and found that RNA isolation rates were consistent provided the tissue disruption and homogenisation steps were followed (described on page 17, “Results and Analytical Methods” subsection “RNA Extraction for RT-qPCR”, figure 6a): “SSBM constructs are disrupted and homogenised prior to extraction of RNA for qPCR analysis, by following a process optimised for these constructs. Disruption involves vortexing samples, without the anchors, with glass beads, then by repeated aspiration using a syringe and needles. Disrupted samples are homogenised using QIAShredders (cat no.: 79656; supplier: QIAGEN), with the resultant sample ready for RNA extraction.” I am not convinced that Fig. 7 suggests the presence of osteocytes in the tissue through a single podoplanin-positive staining in the absence of controls as this protein is also expressed on other cell types. IF staining should include other accepted bone-specific or bone maturation makers. This could be easily checked and should be considered. Author Response: We recognise the reason for the reviewer’s comment. The images in figure 7 were included to evidence that immunostaining of the constructs is possible rather than to draw conclusions about cell differentiation. In this manuscript we make the case that this model is capable of long-term culture of osteoblasts and their differentiation towards osteocytes. In response to the reviewer’s comment (and to a similar comment from reviewer 1), we have amended the language within the manuscript to clarify that this method supports the long-term culture of osteoblasts and their differentiation towards osteocytes, but we stop short of stating that mature osteocyte formation occurs. Of note, images of mature osteocytes within SSBM constructs have been previously published in Iordachescu et al. 2018, which is referenced frequently throughout the manuscript. Given that the aim of the current manuscript was to provide a detailed and practical methodology for a previously published method (albeit with a different cell source), we consider that these previously published images are sufficient. Importantly, while using XRF is a nice idea, demonstrating calcium and phosphorous within the SSBM constructs does not make this a “bone model”. To this end, a major limitation of the present methodology is that no biomineralization has been shown, yet, while the title suggests a “bone model” was developed. Stating that “mineral distribution and maturation appears heterogeneous as found in in vivo bone tissue” does not really convince me. Please provide further evidence. Author Response: We recognise the reviewer’s concern that a single analytical method is not sufficient to ascertain whether the mineralisation that occurs here is true biomineralisation. However, the intention of this paper is to provide a methodology for creating SSBMs as well as indicating the analytical methods by which they can be characterised biologically and structurally. Additional characterisation using Ramen spectroscopy has previously been published (Iordachescu et al. 2018), strengthening the evidence that biomineralization occurs in the SSBM." } ] }, { "id": "176191", "date": "08 May 2024", "name": "Sabrina Ehnert", "expertise": [ "Reviewer Expertise Bone models and bone metabolism in vitro" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 methodology is provided in sufficient detail - except for two points:\nHow often can/should the Trypsin/EDTA treatment be done? This is a bit unclear in the description.\n\nCentrifugation speed should be given in G (not max. centrifugation speed - e.g. ENA isolation).\n\nThe conclusion is a bit ambigious as using, for example, osteosarcoma cells or including osteoclast like cells may not be easily done as these cells have strong proteolytic activity and thus may dissolve the constructs.\n\nAre the 3Rs implications of the work described accurately? Partly\n\nAre a suitable application and appropriate end-users identified? 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": [ { "c_id": "11977", "date": "10 Jul 2024", "name": "Amy Naylor", "role": "Author Response", "response": "Reviewer 3: The manuscript is well written and the methodology is provided in sufficient detail - except for two points: How often can/should the Trypsin/EDTA treatment be done? This is a bit unclear in the description. Response: We thank you for highlighting this missing information and now added the following to this section (5. Maintenance of constructs): “A trypsin wash is recommended to be performed at least 1 week after cell seeding, or once contraction of the fibrin has been established. Repeat the trypsin wash 2 weeks later, however, do not perform trypsin washes once osteogenic conditions have been introduced as this will impact mineralisation.” Centrifugation speed should be given in G (not max. centrifugation speed - e.g. ENA isolation). Response: We agree, thank you for noting this omission – it has now been rectified: ”(2 min; 13 000 G) using a microcentrifuge” The conclusion is a bit ambigious as using, for example, osteosarcoma cells or including osteoclast like cells may not be easily done as these cells have strong proteolytic activity and thus may dissolve the constructs. Response: In our opinion, the addition of additional cell types to the constructs will provide valuable insight into their activity in the context of a bone organotypic matrix and mature osteoblasts, indeed this is something that we, and our collaborators, are currently exploring. We recognise that there may be challenges in the development of the assay in this direction and have therefore softened the original sentence to reflect this: “There is potential for this assay to be adapted to incorporate mixed cell populations to study cell-cell interactions and we encourage further development along these lines”." } ] }, { "id": "169248", "date": "13 May 2024", "name": "Michelle A. Lawson", "expertise": [ "Reviewer Expertise Myeloma-induced bone 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\nFinlay et al. describe a method to produce a 3D, self-structuring bone model that uses a biocompatible scaffold to support the differentiation of osteoblasts to osteocytes and the production of a complex collagen-rich mineralised matrix using hFOB 1.19 cells.\nRegarding the title, since previous bone models have limited osteocyte culture within them, should this key component not be highlighted in the title? For example, would this not be more suitable title “A three-dimensional, self-structuring bone model that uses a biocompatible scaffold to support the differentiation of osteoblasts to osteocytes and the production of a complex collagen-rich mineralised matrix”?\nIn the abstract and introduction, it’s mentioned that the SSBM method can be readily adapted to accommodate osteoblasts derived from a number of different species, from primary osteoblasts isolated from vertebrate bone samples or from cell lines, but you don’t say which until p13 Table 2. These should be mentioned much earlier either in the abstract and/or the introduction. It’s also not clear if all the data shown is using the hFoB 1.19 cells or other cells too.\nThe introduction is clear and describes the limitations of previous bone models and ethical issues of using animal models.\nThe methods describe very detailed, point by point steps with figures on how to set up each of the 5 aspects of the model, including top tips in each step and cell seeding densities depending on species and cell type. However, for the 1 and 2 weeks post cell seeding trypsin wash (p14) it is less clear why the cells attached to the fibrin matrix will not also be detached by the trypsin. Is it because they are already enrobed in the matrix by then?\nThe resulting scaffolds and analysis techniques are also described in a lot of detail along with a troubleshooting table. The culture of primary human osteoblasts is also mentioned but the paper provides no evidence of their use in this novel 3D in vitro bone model. Although not surprisingly patient primary cells will vary between samples in these models, so to demonstrate the model preparation, production, and maintenance the hFoB 1.19 cells are less variable, however, it would be good to see some data on the use of primary patient cells in the SSBM since the authors have stated they can be used.\nMinor points:\nFigure 1 would be better placed in the methods section rather than the introduction.\n\nIn the keywords “in vitro” should be written in italics.\n\np5 in the methods text B-TCP needs to be written out in full.\n\np12 in this calculation (X mg of BSA = X mL of F - 12K medium) F-12K is no written correctly please amend.\n\nP 15 Figure 4, what are the actual cells seeded onto the fibrin hydrogel in these images?\n\nP15 Figure 4Cii says “Calcium phosphate anchor within red dotted ring”, but is should say red dotted area.\n\nP19 Figure 7 (great IF images BTW) but what are the cells used in the SSBM constructs?\n\nTable 5 is not mentioned in the main text.\n\nAre the 3Rs implications of the work described accurately? Partly\n\nAre a suitable application and appropriate end-users identified? 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? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "11978", "date": "10 Jul 2024", "name": "Amy Naylor", "role": "Author Response", "response": "Reviewer 4 Finlay et al. describe a method to produce a 3D, self-structuring bone model that uses a biocompatible scaffold to support the differentiation of osteoblasts to osteocytes and the production of a complex collagen-rich mineralised matrix using hFOB 1.19 cells. Regarding the title, since previous bone models have limited osteocyte culture within them, should this key component not be highlighted in the title? For example, would this not be more suitable title “A three-dimensional, self-structuring bone model that uses a biocompatible scaffold to support the differentiation of osteoblasts to osteocytes and the production of a complex collagen-rich mineralised matrix”? Response: We agree, and have now amended the title to: “A detailed methodology for a three-dimensional, self-structuring bone model that supports the differentiation of osteoblasts towards osteocytes and the production of a complex collagen-rich mineralised matrix” In the abstract and introduction, it’s mentioned that the SSBM method can be readily adapted to accommodate osteoblasts derived from a number of different species, from primary osteoblasts isolated from vertebrate bone samples or from cell lines, but you don’t say which until p13 Table 2. These should be mentioned much earlier either in the abstract and/or the introduction. It’s also not clear if all the data shown is using the hFoB 1.19 cells or other cells too. Response: We have found that this method supports osteoblasts from all species and sources that we have trialled. We use hFOB1.19 throughout the manuscript for simplicity and have noted this now in the abstract to ensure clarity for the reader. Abstract: “Osteoblastic cells (hFOB1.19) are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors.” In addition, we state at the beginning of the methods section which cell source has been used throughout this manuscript. We have now added an additional reference to table 2, so that the reader is directed to this information earlier: Paragraph 2, Methods: “The figures included here were generated using the human osteoblastic cell line, hFOB 1.19 (RRID: CVCL_3708) – selected based on their homogenous human origin, as well their stable karyotype, 21 but the SSBM method can be readily adapted to accommodate osteoblasts derived from a number of different species, from primary osteoblasts isolated from vertebrate bone samples or from cell lines. We have used primary human osteoblasts and the human osteoblast cell line, hFOB 1.19,22 with similar success (data not shown – recommended seeding densities are given in table 2).” The introduction is clear and describes the limitations of previous bone models and ethical issues of using animal models. The methods describe very detailed, point by point steps with figures on how to set up each of the 5 aspects of the model, including top tips in each step and cell seeding densities depending on species and cell type. However, for the 1 and 2 weeks post cell seeding trypsin wash (p14) it is less clear why the cells attached to the fibrin matrix will not also be detached by the trypsin. Is it because they are already enrobed in the matrix by then? Response: The reviewer is right to note that contact with trypsin is likely to affect the attachment of all cells to their matrix. Fortunately, the SSBM constructs sit proud of the tissue culture plastic by virtue of their attachment to the anchors. By using a relatively small volume of trypsin solution (0.5 ml) compared to the usual volume of 3.5 ml, detachment of the tissue culture plastic-adherent cells without much contact with the construct itself is possible: “2. Add 0.5 mL of 2X Trypsin-EDTA to each well and incubate at 37 °C for 3 mins. Gently tap the sides of the plate and add 1.0 mL cell culture media” It is reasonable to assume that some detachment of cells on the underside of the construct may occur, however given that this trypsinisation process is performed only infrequently and only during the early stages (first 4 weeks only) of SSBM formation, this does not have long term implications for the analysis timepoints (8 and 12 weeks). The resulting scaffolds and analysis techniques are also described in a lot of detail along with a troubleshooting table. The culture of primary human osteoblasts is also mentioned but the paper provides no evidence of their use in this novel 3D in vitro bone model. Although not surprisingly patient primary cells will vary between samples in these models, so to demonstrate the model preparation, production, and maintenance the hFoB 1.19 cells are less variable, however, it would be good to see some data on the use of primary patient cells in the SSBM since the authors have stated they can be used. Response: We agree with the reviewer that detailed assessment of the behaviour of different cell types within the SSBM would provide interesting data, however such characterisation is beyond the scope of this paper, which simply seeks to provide a detailed methodology to enable other groups to use and adapt the model for their research interests. We trust that the seeding densities provided for the cell types that we have trialled (table 2) will be useful in supporting researchers in establishing the model in their labs with their cell type of interest. Minor points: Figure 1 would be better placed in the methods section rather than the introduction. Response: Whilst we can see the logic in the reviewer’s statement, we first reference figure 1 in the introduction, therefore we have decided to leave it where it is in the manuscript. In the keywords “in vitro” should be written in italics. Response: Thank you for this observation – we have made the change. p5 in the methods text B-TCP needs to be written out in full. Response: Agreed. We have now made this addition. p12 in this calculation (X mg of BSA = X mL of F - 12K medium) F-12K is no written correctly please amend. Response: Now corrected. P 15 Figure 4, what are the actual cells seeded onto the fibrin hydrogel in these images? Response: We have now referred to the use of “hFOB1.19” to all figures as appropriate. P15 Figure 4Cii says “Calcium phosphate anchor within red dotted ring”, but is should say red dotted area. Response: Now amended. P19 Figure 7 (great IF images BTW) but what are the cells used in the SSBM constructs? Response: Thank you! Now dealt with as above – hFOB1.1.9 added to all relevant figures. Table 5 is not mentioned in the main text. Response: Thank you for spotting this. Underneath the title “Troubleshooting” we have now added the following sentence: “Potential challenges experienced during SSBM generation are describe in Table 5, along with their possible causes and solutions.”" } ] } ]	1	https://f1000research.com/articles/12-357
https://f1000research.com/articles/13-794/v1	12 Jul 24	{ "type": "Systematic Review", "title": "Antecedents of entrepreneurial networking behavior and its impact on business performance - a systematic literature review", "authors": [ "Sheetal Singh", "Dr Savitha Basri", "Sheetal Singh" ], "abstract": "Purpose The purpose of this paper is to review the existing research on the antecedents of entrepreneurial networking behavior namely motivational factors and personality factors and its impact on business performance.\n\nDesign/methodology/approach This study employs a systematic review methodology, adhering to PRISMA guidelines.. Using the SCOPUS database, the search involved Boolean operators to narrow down relevant articles published in English between 2000 and 2024. Following a meticulous screening process, 32 articles were selected for review after removing duplicates and excluding non-English articles. Methodological quality assessment ensuring focused research questions, precise subject selection methods, representative samples, and reliable measurement instruments was carried out.\n\nFindings The results demonstrate that entrepreneurs who are self-reliant and extroverted have fewer partners, however, these traits positively impact networking activities. The Big Five personality traits predict business creation and success of ventures. Desire for financial gain, risk-taking propensity, self-confidence, and the need for achievement influences networking behavior.\n\nImplications The insights given in this paper can be used for establishing valuable connections, investing in resources, and preparing effective strategies for businesses. Policymakers who aim to promote entrepreneurial behavior among experienced individuals must emphasize the motivations for starting a business and the role of personalities in harnessing and leveraging individual entrepreneurial expertise.\n\nOriginality The existing literature on antecedents of entrepreneurial networking Behavior and the relationship of these antecedents together with business performance is limited. Further, this review article also offers avenues for future researchers.", "keywords": [ "Entrepreneurship", "Motivation", "Networking behavior", "Personality", "Social networking" ], "content": "1. Introduction\n\nThe start-up ecosystem in India is the third largest in the world after the USA and China (Economic Survey 2022-2023), and the number of start-ups increased significantly over the decade with the support of several government initiatives such as streamlined procedures, the granting of tax breaks, access to affordable internet services, and an enormous domestic market. However, 90% of start-ups fail within the first five years as a consequence of a lack of innovation, dynamism, or poor product-market fit, lack of leadership, skills, or expertise, inadequate funds, or even an absence of mentorship (Kalyanasundaram, 2018; Kalyanasundaram et al., 2020). In this scenario, the social networking of an entrepreneur can lead his or her business to the next level.\n\nSocial networking is crucial for connecting people in the age of social media and for gathering information, resources, and knowledge. Through networking, people and organizations can interact with others in the sector, exchange knowledge, and discover fresh perspectives (Gloor et al., 2018). Additionally, it might lead to prospects for funding, partnerships, and employment. Attending conferences, taking part in business events, joining professional groups, and making connections on social networking sites such as LinkedIn are just a few ways to build a network. It is important to develop and maintain relationships over time since networking involves more than just making connections, it is also about building long-lasting relationships. While performing social networking, entrepreneurs are often driven by their goals and aspirations, and it is the motivational factor that fuels their determination to succeed (Abdallah et al., 2022). These factors can include personal ambition, a desire for financial success, the need for autonomy and independence, and the gratification derived from overcoming challenges. Research has shown that highly motivated entrepreneurs tend to have higher levels of persistence, resilience, and commitment to their business goals (Murnieks et al., 2020). Similarly, understanding the role of the personality of an entrepreneur for social networking also plays a crucial role in determining business performance (Di Stefano et al., 2023; Gloor et al., 2018). Research has shown that certain personality traits have a significant impact on an entrepreneur’s ability to succeed and perform well in their business ventures. These personality factors include traits such as extraversion, conscientiousness, openness to experience, emotional stability, and risk propensity (Martin et al., 2016).\n\nThe current review attempts to learn more about the antecedents of entrepreneurial networking behavior and its impact on Start-up performance. It also provides some suggestions for further study in this area. Many scholars have conducted systematic reviews of evidence on networking behavior, but a systematic review of the antecedents of networking behavior such as – motivational factors and personality factors, and their effects on business performance has not been conducted. Therefore, it is vital to examine existing research on the causes and consequences of networking behavior. The flow of this review article includes the literature review and its theoretical support, the methods used to identify all relevant articles, findings followed by discussions, suggestions for future research, and conclusions.\n\n\n2. Literature review\n\nSocial networking theory explains the complexities of networking by describing how social systems work among interconnected components (Burt, 1997; Hatala, 2006). It is extensively used for measuring interpersonal dimensions of human interactions that explain how individuals choose different people for particular jobs (Schultz-Jones, 2009). A network is a social structure that is larger than the sum of its parts and is composed of interpersonal relationships between a group of people (Carter and Evans, 2006). Networking, in this context, is the process of developing, maintaining, and activating network relationships to improve the flow of information, capital, power, and friendship through these interactions (Carter and Evans, 2006). In networking relationships, there may be exchanges of friendship, power, information, or goods as well as communication for mutual understanding and reciprocity to ratify trust. Interacting with others increases a person’s likelihood of gaining financial advantages, developing reputation, and establishing economic legitimacy (Shane and Cable, 2002). Networking behavior is related to establishing close proximity to f business partners from whom an entrepreneur acquires business opportunities. Strong relationships are characterized by high level of trust, which enables social interactions and coordination. Weak ties, on the other hand, help to identify opportunities and are positively associated with benefits for the organization (Elfring and Hulsink, 2003). More networking does not result in a large network, it depends on the networking talent of the founders and national cultural circumstances (Baron, 2007). The size, diversity, density, openness, and stability of the entire network can be considered as the composition of a network (Carter and Jones, 2006).\n\nMotivational factors are closely related to business growth and are consistent with prior research in social psychology and applied psychology (Shane et al., 2003). According to Sharafizad and Coetzer (2016), the motivation for beginning a firm can affect business strategy and operations while practicing networking. Men and women network differently, motivated by financial motivation, risk-taking, and self-confidence, with men scoring higher than women and having a stronger desire to build a firm (Carter and Jones, 2006; Coleman, 2007). The need for achievement is also considered the main motivation for growing the business. The desire for independence is another motivational factor that encourages business owners to be their bosses, which has a favorable impact on the growth of their business. The desire for wealth has also been classified as a pull factor, although it may not be the driving force in the beginning (DeMartino and Barbato, 2003; Alstete, 2003; Rosa and Dawson, 2006). In regard to wealth as a motivating factor, there are certain gender disparities (Demartino and Barbato, 2003; Wilson et al., 2004). Baum et al. (2004) discovered that a diverse but interconnected set of motivational qualities and notions (along with talent) influence the performance of businesses. Furthermore, motivational factors can influence an entrepreneur’s decision-making process and ability to take risks. Entrepreneurs who are motivated to achieve success are more likely to set ambitious goals for their businesses, seize entrepreneurial opportunities, and actively pursue strategies for expansion and innovation (Ismail, 2022). Entrepreneurial action can be influenced by the networking style of an entrepreneur, but this action can be changed according to multiple logics, including causality and effectuation approaches (Sarasvathy, 2001; Fisher, 2012). Entrepreneurs believe that being socially enabled can benefit not only themselves but also their businesses. The greater the strength of cause or effect, the more motivated entrepreneurs are to participate in networks, particularly when evaluating the benefits to their businesses.\n\nPersonality may have a large impact on social networks and business success (Jack et al, 2008). The Big Five personality traits are often considered the most comprehensive and accurate specifications of a personality (Holt et al., 2007; McCrae, 2011). Extraversion, agreeableness, openness to experience, conscientiousness, and neuroticism are the five basic elements that this model uses to determine personality. These personality traits are relevant for business studies since they reveal actions that are indicative of entrepreneurial ability (Holt et al., 2007; Obschonka et al., 2012; Zheng et al., 2010). The Big Five personality model is useful for forecasting entrepreneurial business performance (Martin et al., 2016). Several studies have found that higher levels of extraversion are positively associated with business performance. More extroverted entrepreneurs tend to be outgoing, sociable, and assertive, which can help them in networking, building relationships with customers and stakeholders, and confidently promoting their business (Baluku et al., 2016; Kerr et al., 2018). Conscientiousness is another personality trait that is positively associated with business performance among entrepreneurs. Entrepreneurs who are highly conscientious tend to be diligent, organized, and responsible, which can contribute to better planning, time management, and task completion (Jawabri, 2020). Openness to experience has been linked to business performance among entrepreneurs. Entrepreneurs who are open to new ideas, experiences, and opportunities are more likely to adapt and innovate in a rapidly changing business environment (Kerr et al., 2018). This can lead to a competitive advantage, as they are more willing to explore new markets, products, and strategies. Emotional stability, or low neuroticism, is also important for business performance among entrepreneurs. Emotionally stable entrepreneurs are better able to handle stress, setbacks, and challenges that come with running a business. They are more resilient and capable of maintaining a positive mindset, which can help them navigate difficult situations and make sound decisions (Kerr et al., 2018; Smith, 2013). Additionally, risk propensity influences entrepreneurial intentions but not necessarily entrepreneurial performance (Baluku et al., 2016). This resilience enables them to stay focused and motivated, make rational decisions, and maintain a positive mindset, even in difficult circumstances.\n\n\n3. Methods\n\nThis systematic review of quantitative and qualitative studies was designed and implemented following the PRISMA framework (Moher et al., 2009). The researchers used the PRISMA model (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) to establish the structure of the study selection for review. After removing duplicates, 3879 publications were retrieved from the SCOPUS electronic databases. Initially, the titles and abstracts of all studies were screened and assessed against the inclusion and exclusion criteria to determine which were relevant to the review. Overall, 32 out of 230 studies were included in the analysis.\n\nThe selection process is illustrated in the PRISMA flow chart presented in Figure 1.\n\nThe SCOPUS database is used for a systematic search of relevant studies. The search strategies and terms were tailored to each database individually. Searches were performed by combining search terms using the Boolean operators AND, OR, and NOT. Searches were limited to peer-reviewed articles in English languages published between 2000 and 2024 to obtain an updated view of the research topic and to avoid overlapping results with the previous review. The iterative Booleans were utilized in various combinations in the search strategy namely, ‘networking behavior AND business performance’, ‘motivation AND business performance, and ‘personality AND business performance’, Microsoft Excel was used to create a data extraction sheet to reduce human error bias. Initially, 3951 papers were identified, and after determining duplication, 72 papers were considered for further screening. From the total of 230 studies, 32 studies were included for screening based on the research questions and their accessibility. Both longitudinal and cross-sectional research articles were chosen from the years 2000 to 2024, and these articles are mainly based on the English language.\n\n230 articles remained for the title and abstract review (Figure 1). Articles retrieved based on abstracts were excluded from further analysis if they were not in English and irrelevant to the research questions. Reviews, editorials, and discussion papers also exclude the inclusion criteria. The researchers read the full texts of these articles to evaluate their eligibility for the review, resulting in the exclusion of a further 198 articles. The reason for exclusion of the studies did not address the research questions. Researchers screened every article independently. The 32 articles remaining after this process were selected for inclusion, and the researchers manually screened their references to identify additional relevant articles.\n\nThe final data consisted of 32 quantitative and qualitative studies. All the studies included in the review addressed focused questions using appropriate research methods. Furthermore, the methods used to choose subjects has been clearly described. All the studies used representative samples and trustworthy measurement instruments.\n\nSpecifying information about the publication, the purpose of the study, study subjects, study context, and significant findings reported in each article were extracted. The articles were analyzed by narrative synthesis (Popay et al., 2006). Finally, only those 32 research papers that met the eligibility requirements were included in the study.\n\n\n4. Overview of the reviewed studies\n\nTable 1 shows the selected articles considered for the literature review in table format (refer to extended data table 1).\n\n\n5. Findings and discussion\n\nMost of the studies used a constructivist paradigmatic approach that was congruent with the methodologies used including case studies, narrative inquiry, ethnography, mixed-methods, and grounded theory. The majority of the studies have used a quantitative data approach to examine and comprehend entrepreneurs’ perceptions and actual networking behavior. The findings of these studies are significant for various reasons. It was found that most of the studies have been carried out in countries namely India, Slovenia, Uganda, Croatia, Germany, Portugal, and China. This may be because of the promotion of entrepreneurship as a tool for economic growth and unemployment reduction in developing nations, where entrepreneurs can access government incentives for their businesses to grow. The summary of review is given in Table 1.\n\nFurthermore, it was found that the majority of studies used self-rating techniques, such as questionnaires which would have been biased toward some entrepreneurs because they might have given more weight to their positive attributes than to those that were essential for business success. Additionally, regarding personality as an antecedent to networking behavior, the majority of studies have examined only the Big Five traits to date, completely ignoring additional personality factors such as MBTI and nonpersonality factors namely cognition, knowledge, skills, and intelligence. The study sample sizes ranged from 50 to 3384 people. Critical events, content analysis, mixed approaches, embedded case studies, virtual experimental procedures, and in-depth personal interviews were all used in these studies, in addition to surveys. A small percentage of studies have used mixed techniques, with the majority of studies conducting only quantitative or qualitative research. The use of the mixed methods approach, which analyzes the evidence based on the current situation, may therefore be essential.\n\nThe process of building, cultivating, and mobilizing network links is known as networking. It includes the exchange of information, money, strength, and communication that occurs as a result of these ties (Carter and Evans, 2006). Networks help in seeking information and advice, enriching internal resources, competing in flexible marketplaces, and creating advanced products (Wiklund et al., 2009). Carter and Evans (2006) reported that large and complicated networks are more likely to provide business potential, increase problem-solving opportunities, and provide businesses with a greater chance of succeeding. Connecting with like-minded people, obtaining access to expertise and information, increasing learning, and building a sense of belonging and legitimacy are all major benefits of formal networking. Those who are interested in the success of a business use many networking practices (Weber, 2007).\n\nMotivational factors are closely related to business growth and are consistent with prior research in social psychology and applied psychology (Shane et al., 2003). According to Sharafizad (2019), networking should not be viewed as a simple stand-alone commercial activity, but rather as a complicated process that is influenced by the entrepreneur’s perceptions, and these are the reasons for founding the company first. The need for achievement is also considered the main motivation for growing the business. The desire for independence is another pull factor that encourages business owners to be their bosses, which has a favorable impact on the growth of their business. The desire for wealth has also been classified as a pull factor, although it may not be the driving force in the beginning (DeMartino and Barbato, 2003; Alstete, 2003; Rosa and Dawson, 2006). Entrepreneurs are compelled to generate money, particularly in the development stage (Alstete, 2003). In regard to wealth as a motivating factor, there are certain gender disparities (Demartino and Barbato, 2003; Wilson et al., 2004). Baum et al. (2004) discovered that a diverse but interconnected set of motivational qualities and notions (along with talent) influence the performance of businesses. Entrepreneurial action can be influenced by the networking style of an entrepreneur, but this action can be changed according to multiple logics, including causality and effectuation approaches (Sarasvathy, 2001; Fisher, 2012). While causal action stresses the choice of means for achieving the desired effect, effectual action highlights the potential consequences that can be achieved using the available means (Sarasvathy, 2001). While entrepreneurial behavior emerges from commitments created in networks and connections from an effectuation perspective, networks can become a method for causal entrepreneurs to achieve the desired effect; entrepreneurs can choose whether to join these networks or not (Fabris, 2021). Entrepreneurs believe that being socially enabled can benefit not only themselves but also their businesses. The greater the strength of cause or effect, the more motivated entrepreneurs are to participate in networks, particularly when evaluating the benefits to their businesses.\n\nThe Big Five personality traits are often considered as the most comprehensive and accurate specifications of a personality (Holt et al., 2007; McCrae, 2011). Extraversion, agreeableness, openness to experience, conscientiousness, and neuroticism are the five basic elements that this model uses to determine personality. These personality traits are relevant for business studies since they indicate actions that are indicative of entrepreneurial ability (Holt et al., 2007; Obschonka et al., 2012; Zheng et al., 2010). Personality may have an enormous impact on social networks and have a direct impact on consequences (Jack et al, 2008). Despite being a major indicator of entrepreneurial success, start-up capital accounted for only 2% of that success (Baluku et al, 2016). The Big Five personality model is useful for forecasting entrepreneurial outcomes (Baluku et al., 2016). The most important indicators of entrepreneurial success are agreeableness and extraversion. Agreeableness has an impact on the strength of network interactions. The findings of this study indicate that there are numerous components of a person’s social network, which are frequently characterized by a person’s personality. Some people may find it easier to form distinct social bonds and moderate the strength of the interactions than others (Baluku et al, 2016).\n\nNetworking is a personal skill that entrepreneurs, particularly start-ups, require. A start-up can establish long-lasting relationships if the founder has effective communication skills and is well-known in the business world. As a result, business networking is critical for both new and established businesses. Networking gives business owners a competitive edge and gives them access to resources. The greatest outcome for entrepreneurs can be to access business opportunities, informational resources, more funds, and a greater consumer base which can be used to build and utilize partnerships with external stakeholders and organizations. (Albourini et al., 2020) found that by cultivating external and internal contacts, entrepreneurs can improve the growth rate of their start-ups. Networking behavior leads to cultivating and nurturing relationships with other individuals and promoting future projects (Forret and Dougherty, 2004). A rise in the customer base and better access to capital and information result from networking (Albourini et al., 2020; Jack, 2005).\n\n\n6. Summary and conclusion of the study\n\nAlthough empirical research has dominated the field and covers a wide range of studies, there is no commonly acknowledged framework. This extensive research on the antecedents of entrepreneurial networking behavior and its impact on business performance has provided valuable insights into the complex dynamics that underlie the success of start-ups, particularly in countries such as Slovenia, Uganda, Sri Lanka, Germany, Portugal, and China. The findings from these studies have shed light on several critical aspects of networking behavior, personality traits, and their implications for entrepreneurial success. Motivational factors, such as the desire for financial gain, risk-taking propensity, self-confidence, and the need for achievement, play a crucial role in driving networking behavior among entrepreneurs. Understanding these motivations can help entrepreneurs align their networking efforts with their goals and strategies. The Big Five personality traits—extraversion, agreeableness, openness to experience, conscientiousness, and neuroticism—are significant predictors of entrepreneurial success. Entrepreneurs should recognize and leverage their unique personality traits to enhance their networking effectiveness. The frequency, reciprocity, and social interactions within a network are essential elements that can determine the quality and effectiveness of entrepreneurial networking. Entrepreneurs should focus on building meaningful interactions that foster trust and cooperation. Networking can lead to various positive outcomes, including access to business opportunities, information, funding, and a broader consumer base. Entrepreneurs who effectively cultivate and mobilize their network connections are more likely to achieve their business goals. Therefore, entrepreneurial networking is a multifaceted process influenced by motivations, personality traits, network structure, content, and interactions. Entrepreneurs who understand these factors and strategically harness the power of their networks are better positioned for success in the competitive world of start-ups.\n\nWhile these studies have provided valuable insights, there are still numerous avenues for future research. Most of the studies have used cross-sectional analysis, and future studies should undertake a longitudinal study to examine the evolving roles of the focal constructs in the model. There are very few studies adopting a qualitative approach for a specific industry, population group, or context. Therefore, a more qualitative approach may prove very insightful for understanding new industry segments and economic, and sociocultural contexts. The past working experience and cognitive abilities of entrepreneurs can also be studied to determine the differences in the networking behavior of naive and experienced entrepreneurs. Exploring the networking behavior of entrepreneurs in developed nations, considering additional personality factors beyond the Big Five, examining gender and experience effects on networking, and studying networking behaviors in different geographic settings (rural vs. metropolitan) are all areas that warrant further investigation.\n\nThis research is important for entrepreneurs because networking behavior plays a crucial role in influencing business success. The insights given in this paper can be used for establishing valuable connections, investing in resources, and preparing effective strategies for businesses. Policymakers who aim to promote entrepreneurial behavior amongindividuals who have risk-taking propensity and the need for achievement motivation and emphasize the significance of harnessing and leveraging individual entrepreneurial personality dynamics through social networks.\n\n\nAuthor contributions\n\nSheetal Singh - Substantial contributions to the conception or design of the work, searching articles, conceptualization, first draft of the paper write-up.\n\nDr. Basri Savitha - Reviewing the manuscript critically for important intellectual content, writing the final manuscript, and editing the manuscript.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: networking among entrepreneurs. https://doi.org/10.6084/m9.figshare.25773411 (Sheetal, 2024a).\n\nFigshare: PRISMA checklist for “Antecedents of entrepreneurial networking behavior and its impact on business performance - A systematic literature review”. https://doi.org/10.6084/m9.figshare.25710135 (Sheetal, 2024b).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAbdallah GK, Masurel E, Naudé WA, et al.: Unboxing entrepreneurial motivations in Tanzania: Business-related and personal-related factors. J. Afr. Bus. 2022; 23(1): 60–78. Publisher Full Text\n\nAlbourini F, Ahmad AMK, Abuhashesh M, et al.: The effect of networking behaviors on the success of entrepreneurial startups. Manag. Sci. Lett. 2020; 10(11): 2521–2532. Publisher Full Text\n\nAlstete J: On becoming an entrepreneur: An evolving typology. Int. J. Entrep. Behav. Res. 2003; 8(4): 222–234. Publisher Full Text\n\nAntoncic B, Bratkovic Kregar T, Singh G, et al.: The Big Five Personality-Entrepreneurship Relationship: Evidence from Slovenia. J. Small Bus. Manag. 2015; 53(3): 819–841. Publisher Full Text\n\nBaluku MM, Kikooma JF, Kibanja GM: Does personality of owners of micro enterprises matter for the relationship between startup capital and entrepreneurial success? Afri. J. Busi. Manage. 2016; 10(1): 13–23. Publisher Full Text\n\nBaron RA: Behavioral and cognitive factors in entrepreneurship: Entrepreneurs as the active element in new venture creation. Strateg. Entrep. J. 2007; 1(1–2): 167–182. Publisher Full Text\n\nBaum JR, Locke EA, Smith KG: A multidimensional model of venture growth. Acad. Manag. J. 2001; 44(2).\n\nBaum JR, Locke EA: The relationship of entrepreneurial traits, skill, and motivation to subsequent venture growth.J. Appl. Psych.2004; 89(4): 587–598.\n\nBurt RS: A note on social capital and network content. Soc. Networks. 1997; 19(4): 355–373. Publisher Full Text\n\nCaliendo M, Kritikos AS, Stier C: The influence of start-up motivation on entrepreneurial performance. Small Bus. Econ. 2023; 61(3): 869–889. Publisher Full Text\n\nCarter S, Jones-Evans D: Enterprise and small business: Principles, practice and policy. Pearson Education; 2006.\n\nChell E, Baines S: Networking, entrepreneurship and microbusiness behaviour. Entrep. Reg. Dev. 2000; 12(3): 195–215. Publisher Full Text\n\nColeman S: The role of human and financial capital in the profitability and growth of women-owned small firms. J. Small Bus. Manag. 2007; 45(3): 303–319. Publisher Full Text\n\nDemartino R, Barbato R: Differences between women and men MBA entrepreneurs: Exploring family flexibility and wealth creation as career motivators. J. Bus. Ventur. 2003; 18(6): 815–832. Publisher Full Text\n\nDi Stefano G, Ruggieri S, Bonfanti RC, et al.: Entrepreneurship on Social Networking Sites: The Roles of Attitude and Perceived Usefulness. Behav. Sci. 2023; 13(4): 323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonbesuur F, Boso N, Hultman M: The effect of entrepreneurial orientation on new venture performance: Contingency roles of entrepreneurial actions. J. Bus. Res. 2020; 118: 150–161. Publisher Full Text\n\nElfring T, Hulsink W: Networks in entrepreneurship: The case of high technology firms. Small Bus. Econ. 2003; 21(4): 409–422. Publisher Full Text\n\nFabris J, Aoki VCG, Machado HPV, et al.: Motivations for business networking and the effectuation-causation approach to entrepreneurship. Revist. Ibero-Americ. Estra. 2021; 20(1): 17574.\n\nFarrington SM: Does personality matter for small business success? S. Afr. J. Econ. Manag. Sci. 2012; 15(4): 382–401. Publisher Full Text\n\nFernandes S, Belo A, Castela G: Social network enterprise behaviors and patterns in SMEs: Lessons from a Portuguese local community centered around the tourism industry. Technol. Soc. 2016; 44: 15–22. Publisher Full Text\n\nFerreira AI, Braun T, Carvalho H, et al.: Networking to death: On the dark side of start-ups’ external networking. Int. J. Entrep. Behav. Res. 2022; 28(9): 289–310. Publisher Full Text\n\nForret ML, Dougherty TW: Networking behaviours and career outcomes: Differences for men and women?’. J. Orga. behav. 2004; 25(3): 419–437. Publisher Full Text\n\nFisher G: Effectuation, causation, and bricolage: A behavioral comparison of emerging theories in entrepreneurship research. Entrep. Theory Pract. 2012; 36(5): 1019–1051. Publisher Full Text\n\nFranco M, Prata M: Influence of the Individual Characteristics and Personality Traits of the Founder on the Performance of Family SMEs. Eur. J. Int. Manag. 2019; 13: 41. Publisher Full Text\n\nGilinsky A, Lopez RH, Santini C, et al.: Big bets, small wins? Entrepreneurial behavior and ROI. Int. J. Wine Bus. Res. 2010; 22(3): 238–250. Publisher Full Text\n\nGloor PA, Woerner S, Schoder D, et al.: Size does not matter-in the virtual world. Comparing online social networking behavior with business success of entrepreneurs. Int. J. Entrep. Ventur. 2018; 10(4): 435–455. Publisher Full Text\n\nHatala JP: Social network analysis in human resource development: A new methodology. Hum. Resour. Dev. Rev. 2006; 5(1): 45–71. Publisher Full Text\n\nHolt DT, Rutherford MW, Clohessy GR: Corporate entrepreneurship: An empirical look at individual characteristics, context, and process. J. Leadersh. Org. Stud. 2007; 13(4): 40–54. Publisher Full Text\n\nIsmail IJ: Entrepreneurial start-up motivations and growth of small and medium enterprises in Tanzania: The role of entrepreneur’s personality traits. FIIB Bus. Rev. 2022; 11(1): 79–93. Publisher Full Text\n\nJack MH: Social networks and the cognitive motivation to realize network opportunities: A study of managers’ information gathering behaviors. J. Organ. Behav. 2008; 29(1): 51–78. Publisher Full Text\n\nJack SL: The role, use, and activation of strong and weak network ties: A qualitative analysis. J. Manag. Stud. 2005; 42(6): 1233–1259. Publisher Full Text\n\nJack S, Dodd SD, Anderson AR: Change and the development of entrepreneurial networks over time: a processual perspective. Entre. Regi. Develop. 2008; 20(2): 125–159.\n\nJawabri A: The impact of big-5 model leadership traits on team entrepreneurship: An empirical study of small businesses in the UAE. Manag. Sci. Lett. 2020; 10(3): 497–506. Publisher Full Text\n\nJha P, Alam MM: Antecedents of women entrepreneurs’ performance: An empirical perspective. Manag. Decis. 2022; 60(1): 86–122. Publisher Full Text\n\nJonsson S: Entrepreneurs’ network evolution-The relevance of cognitive social capital. Int. J. Entrep. Behav. Res. 2015; 21(2): 197–223. Publisher Full Text\n\nKalyanasundaram G: Why Do Startups Fail? A Case Study-Based Empirical Analysis in Bangalore. Asian J. Innov. Policy. 2018; 7(1): 79–102. Publisher Full Text\n\nKalyanasundaram G, Sutaram R, Bala MH: Successful vs. Failed Tech Start-ups in India: What Are the Distinctive Features? Asian J. Innov. Policy. 2020; 9(3): 308–338. Publisher Full Text\n\nKedah Z, Osman Gani AM, Sarif SM, et al.: Effects of business Jihad on entrepreneurs’ motivation and performance. Int. J. Entrep. Small Bus. 2019; 38(3): 277. Publisher Full Text\n\nKerr SP, Kerr WR, Xu T: Personality traits of entrepreneurs: A review of recent literature. Found. Trends Entrep. 2018; 14(3): 279–356. Publisher Full Text\n\nKottika E, Özsomer A, Rydén P, et al.: We survived this! What managers could learn from SMEs who successfully navigated the Greek economic crisis. Ind. Mark. Manag. 2020; 88: 352–365. Publisher Full Text\n\nLingappa AK, Rodrigues LLR, Shetty DK: Performance differentials of necessity and opportunity entrepreneurs: Through the lens of motivation to learn and female entrepreneurial competencies. J. Entrep. Emerg. Econ. 2024; 16(1): 159–187. Publisher Full Text\n\nMa Z, Zhu J, Meng Y, et al.: The impact of overseas human capital and social ties on Chinese returnee entrepreneurs’ venture performance. Int. J. Entrep. Behav. Res. 2019; 25(1): 67–83. Publisher Full Text\n\nMandala GN, Verma M, Verma A, et al.: The influence of agricultural farmers’ entrepreneurial behavior on the business performance of dairy farmers in Andhra Pradesh. Univers. J. Agric. Res. 2021; 9(2): 57–61. Publisher Full Text\n\nMartin MB, Julius FK, Grace MK: Does personality of owners of micro enterprises matter for the relationship between startup capital and entrepreneurial success?. Afr. J. Bus. Manag. 2016; 10(1): 13–23. Publisher Full Text\n\nMcCrae RR: Personality theories for the 21st century. Teach. Psychol. 2011; 38(3): 209–214. Publisher Full Text\n\nMhlanga O: Identification of personality traits affecting entrepreneurial performance in the hospitality subsector: A five-factor personality model. Acta Commercii. 2019; 19(2). Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann. Intern. Med. 2009; 151(4): 264–269. Publisher Full Text\n\nMurnieks CY, Klotz AC, Shepherd DA: Entrepreneurial motivation: A review of the literature and an agenda for future research. J. Organ. Behav. 2020; 41(2): 115–143. Publisher Full Text\n\nObschonka M, Silbereisen RK, Schmitt-Rodermund E: Explaining entrepreneurial behavior: Dispositional personality traits, growth of personal entrepreneurial resources, and business idea generation. Career Dev. Q. 2012; 60(2): 178–190. Publisher Full Text\n\nPanicker S, Rajan S: The concept of entrepreneurial ability-evidence from women in MSMEs of Karnataka state. Int. J. Entrep. Small Bus. 2020; 41(1): 1. Publisher Full Text\n\nPopay J, Roberts H, Sowden A, et al.: Guidance on the conduct of narrative synthesis in systematic reviews. A Product from the ESRC Methods Programme Version. 2006; 1(1): b92.\n\nPrajawati MI, Wahjoedi W, Wahyono H, et al.: Definition of success according to women entrepreneur in Malang, Indonesia. J. Asian Finance Econ. Bus. 2020; 7(9): 747–753. Publisher Full Text\n\nRadipere S: The effects of motivation as a start-up factor on business performance. Risk Gov. Control: Financ. Mark. Inst. 2015; 5(1): 52–60. Publisher Full Text\n\nRosa P, Dawson A: Gender and the commercialization of university science: Academic frontiers of spinout companies. Entrep. Reg. Dev. 2006; 18(4): 341–366. Publisher Full Text\n\nSarasvathy SD: Causation and effectuation: Toward a theoretical shift from economic inevitability to entrepreneurial contingency. Acad. Manag. Rev. 2001; 26(2): 243–263. Publisher Full Text\n\nSchultz-Jones B: Examining information behavior through social networks: An interdisciplinary review. J. Doc. 2009; 65(4): 592–631. Publisher Full Text\n\nShane S, Cable D: Network ties, reputation, and the financing of new ventures. Manag. Sci. 2002; 48(3): 364–381. Publisher Full Text\n\nShane S, Locke EA, Collins CJ: Entrepreneurial motivation. Hum. Resour. Manag. Rev. 2003; 13(2): 257–279. Publisher Full Text\n\nSharafizad J: Influence of mentors on networking behavior of small business owners: A conceptual framework. Int. J. Glob. Small Bus. 2019; 10(4): 298–323. Publisher Full Text\n\nSharafizad J, Brown K: Regional small businesses’ personal and inter-firm networks. J. Bus. Ind. Mark. 2020; 35(12): 1957–1969. Publisher Full Text\n\nSharafizad J, Coetzer A: Women business owners’ start-up motivations and network content. J. Small Bus. Enterp. Dev. 2016; 23(19): 590–610. Publisher Full Text\n\nSheetal S: Networking among entrepreneurs. figshare. 2024a; 2024. Publisher Full Text\n\nSheetal S: Networking behavior among entrepreneurs. figshare. Figure. 2024b. Publisher Full Text\n\nSingh S: networking among entrepreneurs. [Dataset]. figshare. 2024a. Publisher Full Text\n\nSingh S: Networking behavior among entrepreneurs. figshare. Figure. 2024b. Publisher Full Text\n\nSkokic V: Motivations and Benefits of Entrepreneurial Network Formation. Int. J. Bus. Manag. 2015; 10(9). Publisher Full Text\n\nSmith A: The Relationship of Personality to Entrepreneurial Performance: An Examination of Openness to Experience Facets.2013. Reference Source\n\nSobaih AEE, Al-qutaish AA, Gharbi H, et al.: The Impact of Owner-Managers’ Personality Traits on Their Small Hospitality Enterprise Performance in Saudi Arabia. J. Risk Financial Manag. 2022; 15(12). Publisher Full Text\n\nUdimal TB, Liu E, Lou M: Network reliance and entrepreneurial performance, the role of external networking behaviour and entrepreneurial orientation: The case of rural farmer-entrepreneurs. Innov. Manag. Rev. 2021; 18(3): 308–330. Publisher Full Text\n\nWang C, Steiner B: Can social capital explain business performance in Denmark? Empir. Econ. 2020; 59(4): 1699–1722. Publisher Full Text\n\nWeber SS: Saving St. James: A case study of farm women entrepreneurs. Agric. Hum. Values. 2007; 24(4): 425–434. Publisher Full Text\n\nWiklund J, Patzelt H, Shepherd DA: Building an integrative model of small business growth. Small Bus. Econ. 2009; 32(4): 351–374. Publisher Full Text\n\nWilson F, Marlino D, Kickul J: Our entrepreneurial future: Examining the diverse attitudes and motivations of teens across gender and ethnic identity. J. Dev. Entrep. 2004; 9(3): 177–197.\n\nWitt P: Entrepreneurs’ networks and the success of startups. Entrep. Reg. Dev. 2004; 16(5): 391–412. Publisher Full Text\n\nYates VA, Vardaman JM, Chrisman JJ: Social network research in the family business literature: A review and integration. Small Bus. Econ. 2022. Publisher Full Text\n\nZakaria N, Ahmad MZ, Abdullah S, et al.: Examining the Role of Social Network on Opportunity Recognition: A Propose Framework. Int. J. Econ. Res. 2017; 14: 303–311.\n\nZheng J, Yang J, Feng H: Personality trait structure of Shanxi business tycoons in the Chinese Ming and Qing dynasties. Int. J. Psychol. Stud. 2010; 2(2): 117. Publisher Full Text" }	[ { "id": "302646", "date": "05 Aug 2024", "name": "Doraiswamy Ashok", "expertise": [ "Reviewer Expertise Management", "Marketing", "and Entrepreneurship" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reviews the existing research on the antecedents of entrepreneurial networking behaviour, namely motivational factors and personality factors and its impact on business performance.\n\nThis study employed a systematic review methodology adhering to PRISMA guidelines. Using the SCOPUS database, the search involved Boolean operators narrowing down relevant articles indexed in English between 2000 and 2024. The researcher has justified the period of review process on all possible materials up to a satisfactory level. The flow of this review article includes the literature review and its theoretical support, the methods used to identify all relevant articles, findings followed by discussions, suggestions for future research, and conclusions. Literature review coverage about networking behaviours, motivational factors and personality factors were mentioned in the highlights of theoretical models.\n\nIn the method session of the article, the model of PRISMA has been demonstrated step by step with the appropriate number of articles reviewed. Thus, the search criteria have proved satisfactory.\n\nThe findings and discussions were made with available insights from reviews used for the study purpose. Over all, the objective of the paper has served the purpose of demonstrating the antecedents of entrepreneurial networking behaviour.\n\n“I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.” Therefore it is approved for further consideration.\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\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] }, { "id": "315625", "date": "27 Aug 2024", "name": "Roshan Raj Bhujel", "expertise": [ "Reviewer Expertise This paper has key implications for policy and practice by highlighting how personality traits and motivations influence entrepreneurial networking and business success.Policy Implications: Policymakers can use these insights to design targeted support programs and incentives that align with traits like self-reliance and motivations such as financial gain", "encouraging more effective networking among entrepreneurs.Practical Implications: Entrepreneurs can apply these findings to tailor their networking strategies based on their personality traits", "while business educators can incorporate these insights into training programs to better prepare aspiring entrepreneurs for success." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Antecedents of Entrepreneurial Networking Behavior and its Impact on Business Performance - A Systematic Literature Review\" by Sheetal Singh and Savitha Basri is well-researched, offering a comprehensive and methodologically rigorous review of the existing literature. The study effectively utilizes PRISMA guidelines, ensuring a thorough and reliable analysis of how personality traits and motivational factors influence entrepreneurial networking behavior and business performance.\nStrengths:\nMethodological Rigor: The authors' systematic review, guided by PRISMA, is commendable for its clarity, thorough search strategy, and rigorous screening process, enhancing the reliability of the findings.\nComprehensive Coverage: The review covers a broad time frame (2000-2024) and focuses on relevant literature, contributing to a well-rounded understanding of the topic.\nKey Findings: The identification of personality traits such as self-reliance, extroversion, and the Big Five traits, along with motivational factors like financial gain and risk-taking, provides valuable insights into entrepreneurial success.\nPractical Implications: The paper offers actionable recommendations for entrepreneurs and policymakers, emphasizing the importance of networking and strategic resource investment.\nOriginal Contribution: By addressing gaps in the literature and suggesting future research directions, the paper adds significant value to the field.\nAreas for Improvement: Clarification of Concepts: A brief, more detailed explanation of how the identified factors interact to influence business performance could further enhance understanding.\nConclusion: This paper is a valuable contribution to the study of entrepreneurial networking behavior and its impact on business performance. The systematic review, comprehensive analysis, and practical implications make it worthy of acceptance.\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/13-794
https://f1000research.com/articles/13-793/v1	12 Jul 24	{ "type": "Review", "title": "Neurotoxicity of nanoplastics: A review", "authors": [ "Adrienne Jia Wen Gan", "Kiat Fatt Chia", "Chooi Ling Lim", "Boon Keat Tan", "Shew Fung Wong", "Soi Moi Chye", "Chee Onn Leong", "Rhun Yian Koh", "Adrienne Jia Wen Gan", "Kiat Fatt Chia", "Chooi Ling Lim", "Boon Keat Tan", "Shew Fung Wong", "Soi Moi Chye", "Chee Onn Leong" ], "abstract": "With the increase in plastic waste in the environment, it is undeniable that humans and most organisms are exposed to plastic particles of various sizes, including nanoplastics (NPs). Humans are at risk owing to various routes of entry, including ingestion, inhalation, and dermal contact. While the toxicity of NPs is still debatable due to the scarcity of resources and research, most studies have concluded that NPs may exert toxicity, which exacerbates their neurotoxicity potential. Earlier studies concluded that NPs can cause oxidative stress, which results in apoptosis of neuronal cells. Some studies have shown that NPs can affect fundamental cell functions by inducing physical stress through deposition. Furthermore, studies on in vivo models exposed to NPs have demonstrated behavioral changes that are presumably due to alterations in acetylcholinesterase activity and neurotransmitter levels. This review discusses studies conducted on the neurotoxic potential of NPs and their effects, which are dependent on several parameters, including size and type of NPs, exposure concentration, duration, and various models at risk of NP exposure. Furthermore, speculations on how NPs are related to neurotoxicity are also discussed.", "keywords": [ "Nanoplastic", "toxicity", "nervous system" ], "content": "Introduction\n\nPlastics (also known as synthetic polymers) have revolutionized modern everyday life, making the world without plastics almost unrecognizable. Plastics are chains of organic subunits (“monomers”) linked through strong covalent chemical bonds. In 1907, Belgian chemist Leo Baekeland developed the first ever true synthetic polymer, Bakelite, marking the commencement of the “Age of Plastics.” Mass plastic production began several years (in the 1940s and 1950s) after the discovery of Bakelite. At that time, plastic utilization became a heated discussion among industries. A book entitled ‘Plastics’ written by Yarsley & Couzens (1941/45) discussed the indispensable benefits of plastics in a wide variety of sectors.1 Since then, the development of plastics has gradually expanded, with at least 15 new classes of polymers being synthesized.2\n\nTo understand the magnitude of the benefits that plastics have introduced in the modern world, it is essential to comprehend the definition of plastics. Generally, plastics polymerize monomers derived from fossil oil, natural gas, or coal. Virgin plastic polymers are extremely rare, and most are synthesized with various chemical additives to enhance their potential. For instance, the addition of carbon and silica reinforces the polymer material.3 Occasionally, various antioxidants and stabilizers are added to plastics to increase their half-life, thereby slowing the process of environmental degradation. Plastics with different compositions and properties can be manufactured using these enhancers. Currently, plastics are grouped into 20 classes based on their grade, variety, and composition.4\n\nThe manufacture of plastics requires extreme precision because the materials to be engineered require high accuracy and specific physical properties. Plastics are developed under certain heat and pressure conditions, and their material properties are adjusted during and/or after manufacturing to attain desired characteristics such as strength and permeability. Furthermore, plastics can be molded into limitless shapes and sizes via rotation, extrusion, compression, blowing, or injection, thereby allowing the development of a vast array of products.5 Although extra effort is needed for their production, plastics remain inexpensive. Thus, they have gradually displaced materials such as rubber, wood, and metal in a wide variety of sectors owing to their unique and versatile properties: electrical, heat, chemical, and light resistance; strong and durable but meltable at specific temperatures; and cost-effective.2\n\nWith the advent of the plastic revolution, these versatile polymers have been mass-produced to accommodate rapid economic growth and urbanization. Statistics reveal that global plastic production has skyrocketed over the past 70 years, from 1.5 million metric tons (Mt) in the 1950s to approximately 367 million Mt in 2020.6\n\nAlthough it is undeniable that plastics are important in the modern world, the increasing usage of these persistent and ubiquitous materials has led to an emerging threat to the environment. Similar to all consumer materials, plastic waste contributes to an increase in municipal solid waste and mostly to urban litter. This is because most plastics generated today are made from nonrenewable and nonbiodegradable petrochemicals, translating to persistent and prolonged plastic litter endurance.2\n\nIn addition, proper plastic waste management (PWM) failed to achieve public acclaim. A study conducted revealed that the practice of PWM from 1980 to 2015 included discarding (55%), recycling (20%), and incineration (25%).7 In the early days, most plastics were discarded through landfill, as the waste volume remained stable without generating significant levels of harmful leachates or gases. However, this method does not promote plastic waste deterioration, as even biodegradable waste, such as paper, requires an extended period to decay because of the lack of moisture and oxygen needed for the biotic process. The decreasing capacity of landfills and persistence of plastics in the environment could pose an issue in the future. Furthermore, a vast majority of commodity plastics are not readily biodegradable. In this context, the lifetime of plastics is highly variable, as degradation depends on their chemical composition and the condition of their immediate environment.8\n\nEven with the development of new PWM methods, the responsibility for the recovery efforts still lies with the public. Inevitably, littering is a critical social issue that requires attention and resources to promote awareness of its grave consequences for the environment. Mismanaged plastic wastes accumulate and contaminate a wide range of terrestrial environments, including freshwater, seashores, the open ocean, and the seafloor.9 A study by Topçu et al. and Thiel et al.10,11 revealed that more than 80% of litter stranded on beaches are plastic-containing materials such as single-use bags and food containers, which are responsible for the massive quantities of plastic debris (~79%) in the environment.12\n\nDespite being persistent recalcitrant materials, plastics ultimately fragment at an extremely slow rate. The term ‘degradation’ refers to the breakdown of polymer chains owing to alterations in their material properties. Plastics can be degraded either biologically (by microbial biodegradation) or environmentally (through wind, rain, and sunlight exposure). Commonly, sunlight is particularly effective in degrading plastic waste owing to solar ultraviolet (UV)-induced photodegradation reactions.13 Tiny plastic fragments are termed according to their sizes: secondary microplastics (MPs) and nanoplastics (NPs), the latter of which is the primary focus of this review.\n\n\nNanoplastics (NPs)\n\nNPs are one of the very persistent contaminants, particularly in marine ecosystems, and are essentially different from bulk-form polymer types.14 The concern about NPs as an emergent contaminant triggered burgeoning research on its sources, effects, and potential hazards, especially to human health. However, this evidence is starkly deficient. First, the definition of NPs has been intensely debated. NPs have been categorized as plastic particles of either < 20 μm,15 < 1 μm in at least one of their dimensions,16 or < 100 nm in size.17 This review adopts a broader approach to NP size by Hartmann et al.18 (1–1000 nm-sized particles).\n\nNPs can be derived from primary or secondary sources. Primary NPs are created by the deliberate production of polymer materials for specific purposes, such as in consumer products such as cosmetics, medicine, and raw materials.19–21 Secondary NPs are commonly mismanaged bulk plastic waste that are fragmented in the environment.\n\nConvincing evidence suggests that the fate of NPs is often associated with the marine environment. Although information on how NPs are measured in aquatic systems is limited, it has been demonstrated that marine environments constitute the final recipients of plastic particles and NPs. For instance, plastics from land-based sources leak into the sea through freshwater and buoyed by the wind, rendering NPs mostly in aquatic environments.22 Thousands of particles/m2 have been detected in marine environments, further supporting this notion.23\n\nIn the marine environment, there are three main sources of NPs: (1) Primary NPs (polymer NPs that are produced deliberately for specific applications such as drug delivery, biomedical products, electronics),22 (2) Secondary NPs (degradation of bulk plastic wastes in the environment via mechanical action, UV photodegradation, hydrolysis, or microbial biodegradation),13 and (3) inadequate disposal infrastructure at Wastewater Treatment Plants (WWTP).\n\nThe first source (primary NPs) originates from nanoparticle-sized plastics produced for specific applications. These include biodegradable nanoparticles, nanospheres, and nanocapsules used in the medical field,24 and plastic particles of approximately 4 μm in size that are incorporated into cosmetic products such as exfoliating scrubs and skin cleaners.25\n\nThe second presumed source is the degradation of macroplastics in the environment. As previously mentioned, the breakdown process occurs because of biotic and/or abiotic processes. Biotic processes include microbial biodegradation, whereas abiotic processes include mechanical effects (wave forces), wind, rain (hydrolysis of polymers), UV radiation, and photooxidation.13 Such processes contribute either solely or jointly to decreasing the size of particles, from macro, micro, and ultimately to nano dimensions.26 The degradation of plastics was successfully demonstrated under the experimental conditions. For example, research by Zhang et al. on the thermal cutting process of polystyrene foam was able to generate nanosized polymer particles with a range between 22 and 220 nm.27 Lambert and Wagner et al. demonstrated that polystyrene coffee cup lids disintegrated into particles of 1.26 × 108 particles/mL with an average size of 224 nm in only 56 days. Overall, it can be concluded that the degradation depends greatly on the size of the initial plastics to achieve nanoscale dimensions.\n\nPlastic fragmentation occurs because of a drastic reduction in the average polymer molecular weight. Polymers consist of a mixture of chains with variable lengths chemically linked by weak bonds (i.e., hydrogen or van der Waals) or physical interactions between chains. The weak interactions contribute to the brittleness of the bond at low-energy levels. In combination with external forces such as friction, plastics can be fragmented easily – a postulation reinforced by a study done by Shim et al., where expanded polystyrene beads disintegrated into micro- and nanosized particles through glass beads and sand mechanical abrasion in a month.28 This experiment mimicked the conditions at seashores or riverbanks, where abrasion by sand particles against plastic litter for a prolonged duration can eventually lead to the formation of NPs. In addition, the combination of UV radiation, elevated temperature, and humidity accelerated the rate of fragmentation. However, the association between physical abrasion and NP formation remains unclear.\n\nPhotodegradation is a widely accepted cause of NP production is photodegradation.29 This is because exposure to oxygen and sunlight initiates polymer fragmentation. Therefore, it can be deduced that the rate of degradation increased with physical abrasion, photodegradation, and the combined effects of both. There are two types of nano-fragmentation: (1) direct fragmentation of macro-or microplastics and (2) further gradual size reduction through degradation.22 The formation of NPs in oceans is based on the second type of nano-fragmentation and is estimated to be slower due to the lack of factors such as oxygen and light.\n\nThe third source of NPs is WWTP. Biosolid and effluent waters containing plastic particles from WWTP may leak into the marine environment without proper disposal methods. Talvitie et al. conducted a comparative study on the concentration of microplastics in wastewater from a WWTP located in the Baltic Sea and in seawater samples. It was shown that the particles per litre were 25 times higher than those of seawater samples.30 Another example of the presence of NP particles in synthetic fibers showed that > 1900 fibers per item could leak into sewage during each washing of clothes.31 Therefore, plastic particles entering the marine environment without proper disposal are a grave concern.\n\nIn most studies, detection and quantification methods for MPs in various environmental samples have been successful. However, analytical methods for detecting NPs in the environment have not yet been established. There are 33 studies on developing established protocols for pre-treatment (digestion and preconcentration), isolating, identifying, and quantifying NPs.18 Unfortunately, most were only able to detect the presence of NPs under laboratory conditions but failed to isolate and quantify environmental NPs from original field samples.\n\nUV-VIS spectrometry, electron microscopy, field flow fractionation (FFF), and dynamic light scattering (DLS) are some techniques used in nanoparticle assessment.32 Presently, it is presumptuous to think that these methods are appropriate for NPs, as each technique has limitations. Shim et al. successfully identified the presence of NPs under mechanical abrasion against plastics using scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS).28\n\nOther analytical methods such as Coulter Counter and nanoparticle tracking analysis (NTA) characterize particles in environmental samples.33 Coulter Counter is a conductivity-based method that measures particles of 0.4 – 1200 μm in diameter using an aperture. This method is highly dependent on the size of the aperture used.34 In this technique, the sample is placed in a weak electrolyte solution to be diluted, and then allowed to pass through the aperture, which is concurrent with an electrical current. As the particles in the sample passed through the aperture, they displaced their own electrolyte and changed the impedance. Alterations in the impedance generate a pulse that is directly proportional to the volume of particles displaced and converted to a signal. This signal was then passed to an instrument to assess and measure the volume and size distribution of the particles.35,36 NTA allows the visualization, measurement, and characterization of particles ranging in size from 30 to 2000 nm.37 This technique uses a laser beam to illuminate a single particle in the sample of interest; the particle size is proportional to the rate of Brownian motion.38 NTA has been previously used in studies by Lamber and Wagner for the degradation of polystyrene.39\n\nIn general, published studies have described a series of procedures before the identification and quantification of NPs in real field samples. These include sampling, pre-treatment, imaging (for identification), and quantification (of its concentration and size distribution). Sampling involves the collection of a portion of real field samples, whereas pre-treatment is a process of retrieving nanoparticles from environmental samples such as freshwater, wastewater, sediments, soils, and food.40,41\n\nAs NPs are highly heterogeneous and can undergo aggregation with organic and inorganic materials, pretreatment is important to separate impurities while retaining the amount and essential properties of the NPs.42 Three types of pretreatments, which are performed solely or jointly, include digestion of the matrix, preconcentration, and separation. The identification of NPs involves chemically identifying whether the nanosized particles are plastic. The methods used are spectroscopy (e.g., SEM-Raman spectroscopy, X-ray photoelectron spectroscopy (XPS),43–46 mass spectrometry (e.g., pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS),47–50 thermal desorption-proton transfer reaction-mass spectrometry (TD-PTR-MS),51 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).52\n\nQuantification of NPs is the final procedure of the analytical methods used to detect NPs in environmental samples. Usually, quantitative data, such as particle size distribution, particle concentration, and mass concentration, are considered for the assessment of the potential risks of NPs to the environment. However, the quantification of NPs has yet to be established and discovered. Some of the methods used include SEM, TEM, DLS, and NTA.18\n\nTo date, only five studies have reported the detection of environmental NPs in field samples. One study isolated, quantified, and identified nanosized plastic particles in seawater samples from the North Atlantic subtropical gyre using ultrafiltration, DLS, pyrolysis, thermal desorption, and thermochemolysis coupled with GC-MS.53 Another study used membrane filtration and TD-PTR-MS to analyze polyethylene terephthalate (PET) in alpine snow samples. The other two studies investigated NPs in sand and agricultural soil samples, respectively, using Py-GC-MS.49,50\n\nDespite the findings discussed here, analytical methods for NPs in field samples remain scarce, suggesting that the occurrence of NPs may be speculative.54 Nevertheless, recent literature has highlighted the presence of nanometric particles produced by degradation. It is assumed that NPs are present particularly on the ocean surface, with intense exposure to sunlight and abundant oxygen. The distributions of NPs depend on their buoyancies; for instance, NPs with higher densities sink and accumulate in sediments. Less dense NPs remain on the surface of the sea and face biofouling, entrain with biological aggregates, or degrade further.55\n\nThe understanding of NPs and their interaction with the environment is vague owing to methodological challenges. Currently, NPs in environmental samples remain largely unquantified. Although NPs have recently evolved to a heated topic in research communities, many characteristics of NPs have been assumed through the data obtained from MPs owing to their similar composition and sources. Some may even assume that the interactions of NPs are analogous to those of MPs because of their similar constituents and sources. However, both plastics are distinguishable based on their size distribution, transport properties, bioavailability, interactions with environmental materials, and potential toxicity towards organisms.\n\nHence, it is essential to define ‘MPs’ and ‘NPs.’ Although studies on NPs have been inconsistent with their exact size threshold compared to MPs, the US National Nanotechnology Initiative defines ‘nano’ as having at least one dimension in the range of 1 to 100 nm. MPs are plastic particles that are less than 5 mm in size. However, the more obvious differences lie in their distinct characteristics. In terms of sedimentation and buoyancy, a study found that NPs have a higher dominance of Brownian motion in suspensions. This is because the NPs are more likely to be homogeneously dispersed. Furthermore, the ability of NPs to heteroaggregate or adsorb with other natural colloids (e.g., natural organic matter (NOM)) is higher because of their small sizes. However, this largely depends on environmental conditions such as porewater pH, ionic strength, and hardness. As NPs heteroaggregate with natural colloids, they affect the environmental fate, as NPs are smaller than most microbes. Heteroaggregates formed with NPs are relatively difficult to discriminate from their plastic components owing to microbial interactions. Additionally, most plastics are chemically modified and NPs have short scales in diffusive transport, which accelerates the release of these additives.\n\nIn terms of biological consequences, the minute size of NPs increases their bioavailability and accessibility in biological organisms through biouptake, translocation, and transportation across membranes. As NPs are approximately the same size as natural proteins, they can pass through biological membranes through passive diffusion or other pathways, such as endocytosis. Thus, increased bioavailability may indicate higher toxicity. Rist et al. conducted a toxicity study of MPs and NPs in Daphnia magna by assessing the feeding rates and reproduction of crustaceans. The results revealed that nano-polystyrene particles caused significantly decreased feeding rates in plants compared to MPs, and also deduced that nanoparticles produce reactive oxygen species after interacting with subcellular components.56 NPs also have a higher surface reactivity than MPs owing to their exposure to specific molecules. This results in a series of consequences for the surface chemistry of biological systems.\n\nIn conclusion, both nano- and microplastics are hazardous to most organisms, including humans, owing to their high specific surface area and hydrophobic nature, which allows them to adsorb easily on certain surfaces.57 Furthermore, their miniature size allows them to act as vectors for both chemical pollutants and pathogens. A study by Wu et al. proved that MPs provide a microhabitat that supports the growth of certain bacteria, thereby increasing opportunistic human pathogen resistance to antibiotics.58 Alarmingly, plastic particles with sizes of MPs or smaller can translocate across biological membranes through endocytic-like mechanisms, which results in cell and/or tissue internalization and adversely affects health. In contrast, plastic particles larger than 100 μm are generally non-toxic.\n\nAs studies on NPs and their toxicity towards human health are limited, the data suggest that NPs are more hazardous to human health than MPs. Jeong et al. and Rist et al. provided factual evidence that smaller MP show higher bioavailability and retention time in the body compartments, which implies higher toxicity levels.59,60 Furthermore, NPs show a higher potential for bioaccumulation in organisms and can pass through biological barriers. In addition, NPs have a higher surface-to-volume ratio and aggregation potential than MPs,61 which allows them to interfere with most biological and chemical interactions in the body.62\n\nAs NPs have only recently commanded the spotlight, it is not surprising that information on the potential effects of NPs is still scarce. In contrast to NPs, the routes for MPs entering the human body have been relatively well studied and thus form the basis for work on NP transport and bioaccumulation. In this regard, NPs can enter the human body through ingestion, inhalation, and skin contact in three ways.\n\nIngestion. The primary route of human exposure to NPs is through ingestion of contaminated food or drinks. In this context, the entry of NPs into food and beverages may be due to the migration of NPs from packaging materials or via the food chain. The initial analysis to verify ingestion as the route of NP internalization was by studying the presence of plastic particles excreted in human stool samples. The study also reaffirmed that humans experience continuous exposure to NPs through the uptake of food and water.63\n\nThe most well-defined route is drinking water, especially in countries where water is consumed directly from pipes. This is because water networks are connected from household to household through plastic-based plumbing. As expected, NPs are produced through long-term, high levels of fluid force on the piping walls, which eventually wind up in consumers’ gastrointestinal (GI) tracts.64 Additionally, packaging can be used as a source of plastics. A study reported that billions of NPs are ingested by consumers from a single cup of tea per plastic teabag.65\n\nCommon food products, such as sugar, salt, bottled water, and aquatic organisms, such as bivalves, fish, and crustaceans, are known to be impregnated with MPs and NPs. However, studies on MP absorption through the diet are more prevalent than those on NPs. For instance, a study on table salt consumption per person (which is higher in China66 than in Europe67) measured MPs that were inadvertently mixed with table salt and showed that 37 and 100 plastic nanoparticles were consumed per person in Europe and China, respectively.66,67\n\nThe presence of MPs and NPs in food, particularly seafood, was reported by the ESPA Panel on Contaminants in the Food Chain (CONTAM). In 2016, they quantified the amount of MPs that entered the human digestive system by extracting, detecting, quantifying, and characterizing plastics using Fourier-transform infrared spectroscopy or Raman spectroscopy. The average number of MPs found in fish is 1-7 particles/g and bivalves is 0.2-4 particles/g, while data on NPs are limited due to technical challenges in analytical methods.\n\nAnother study revealed that MPs and NPs can be ingested by Pacific oyster larvae,68 further supporting the presence of plastic particles in food. The entry of MPs and NPs through this pathway causes pathological changes in the digestive tract, including gut barrier dysfunction, intestinal inflammation, and dysbiosis of gut microbiota.69–72\n\nThe transfer of plastic particles between trophic levels may play a significant role in the presence of small plastic particles in food. Evidently, the human consumption of aquatic organisms can be linked to the food chain, whereby bioaccumulation and subsequent transfer of NPs through trophic levels occur.68,73 Other routes include deposition of NPs in the gills or skin of aquatic organisms.74\n\nThere are three assumptions regarding the fate of NPs after entering the GI tract: whether they remain in the gut lumen, translocate across the gut epithelia into systemic circulation, or are excreted from the body. In comparison to MPs, NPs were able to pass through the pores at paracellular tight junction channels with a maximum size of 1.5 nm.67 Other routes for NPs to enter systemic circulation are through the lymphatic system or phagocytosis by microfold (M) cells in Peyer’s patches.75\n\nTwo studies conducted on the absorption of polystyrene NPs in vivo and in vitro suggested that the oral bioavailability of polystyrene NPs was 10 times higher than that of MPs.76,77 However, the rate of NP absorption varies greatly, and there is no correlation between the size and chemical structure of NPs. The absorption rates of NPs in the GI tract are of great interest because NPs undergo transformation post-ingestion, which in turn affects their absorption rate and ability.\n\nThe heteroaggregation ability of NPs enables them to interact with organic molecules such as proteins, lipids, and carbohydrates.78 Eventually, the particle surface is bound by clusters of proteins, forming a coating known as the “corona”.79 This coating critically affects the biological identity of NPs, including their biodistribution and therapeutic and pathophysiological effects.80 The presence of a corona enhances potential NP toxicity in humans, as protein-coated NPs can interact more easily between tissues and organs than virgin NPs.81 Gopinath et al. revealed that protein-coated NPs led to higher cytotoxicity and genotoxicity in an in vitro study of human blood cells in comparison with bare NPs.82 This could be due to the formation of a biomolecular corona on the surface of NPs, which prevents them from being detected by immune cells and thus persists longer in the circulation.\n\nInhalation. Humans can also be exposed to NPs through unintended inhalation or occupational exposure.83 Airborne NPs originate from either outdoor (rubber tyres, fertilizers, and sea salt aerosols) or indoor environments (synthetic textiles and agriculture). A study revealed that NPs are present in atmospheric fallout.84 Dris et al. showed that most MPs from atmospheric fallout in both urban and suburban areas of Paris were derived from synthetic fiber particles. The atmospheric flux from urban areas is twice the average amount and exceeds that of suburban communities. A further study by Dris et al. indicated that MPs are higher in indoor samples (1-60 fibres/m3) than in outdoor samples (0.3-1.5 fibres/m3).85,86 However, information on the amount, source, and concentration of airborne NPs is limited.\n\nThe entry of NPs into an organism can lead to many health consequences, either through the toxicity of the particle itself and/or its chemical constituents, or by introducing pathogens and parasite vectors.87 Inhaled NPs are destined to be deposited in the lungs, which is a part of the complex network that comprises the respiratory system. Approximately 300 million alveoli facilitate gas exchange via diffusion, encompassing a surface area of approximately 150 m2 and a fine tissue barrier of less than 1 μm.88 Air in the alveoli is in proximity to the flowing blood, as the lumen and alveoli are only separated by an epithelial and endothelial layer.\n\nUnder normal circumstances, foreign particles deposited on the lungs are removed as they enter the respiratory tract or bronchial tract via mucus and cilia. However, fine particles (< 2.5 μm) such as NPs are transported into the alveoli along with air. Mucociliary transport mechanisms are absent in the alveoli; thus, trapped foreign particles are removed via macrophage engulfment and transported back to the bronchial regions for eradication. Some studies have shown that a small proportion of nanoparticles (<0.05%) can be transported into the bloodstream by overcoming the air-blood barrier. Although this circumstance is uncommon and is dependent on the physicochemical properties of the NPs, this suggests that uptake into the lungs is possible at high concentrations.\n\nFurthermore, there are two fates for plastic particles that enter the respiratory system depending on their physical properties (size and density): deposition or penetration deeper into the lungs. NPs with lower densities and smaller sizes can be embedded deep into the lungs, deposited on the alveolar surface, or translocated to other body parts.89,90 The deposition of NPs on alveolar surfaces can trigger clearance through macrophage action and eventually translocate to the circulatory and lymphatic systems.91\n\nSeveral studies have reported that NPs may induce inflammatory responses in the lung tissue. Xu and colleagues demonstrated that internalized polystyrene NPs (PS-NPs) interfere with gene expression in A549 cells, resulting in the induction of inflammation and apoptosis. Cell viability is dependent on the NP concentration and size.92 Another study by Shi et al. investigated the toxicity exerted by PS-NPs on the same cell line in terms of cell viability, oxidative stress, and inflammatory reactions.93 Taken together, the absorption of NPs via inhalation can result in lung damage. Several factors, including hydrophobicity, surface charge, functionalization, and size, affect the rate and ability of NP absorption in the lungs.94 Absorption rates of NPs through the lungs were found to correlate with occupational exposure and comorbidity in in vivo models, such as pulmonary inflammation and cancer.95\n\nGenerally, respirable NPs may trigger a series of reactions such as oxidative stress, inflammation, and genotoxicity in the respiratory, cardiovascular, and nervous systems. However, the available literature on these mechanisms is inconsistent because of variations in the NP sizes used. Because NPs have a large specific surface area, they contribute to the formation of free oxygen radicals or reactive oxygen species (ROS), such as superoxide anions and hydroxyl radicals. Large quantities of ROS generated in cells create havoc on cellular components such as lipids, proteins, and DNA, causing long-term cell damage in the long run. ROS are formed through one of several mechanisms: (1) direct formation on the surfaces of the NPs, (2) triggered by NPs through mitochondrial damage and disruption of balance in the respiratory chain, or (3) triggered by macrophages and neutrophils activated by foreign particles. These radicals trigger inflammation if they are not eradicated immediately via binding to endogenous antioxidants (e.g., Vitamin C) or degraded by antioxidative enzymes.\n\nDermal contact. Another route of NP exposure is through dermal contact. The human body is protected from injuries, chemicals, and microbial agents by the stratum corneum, which is the outermost layer of skin. The stratum corneum consists of hydrophilic lipids, such as ceramide, long-chain free fatty acids, and cholesterol.96 Owing to these properties, the entry of hydrophobic NPs into the body seemed improbable; however, the skin barrier only restricted the entrance of particles with sizes above 100 nm.\n\nThe dermal route comprises contact via cosmetic and healthcare products or NP-contaminated water. The introduction of NPs through direct contact with the skin is uncommon, but they may invade the body through sweat glands, skin wounds, or hair follicles.97 Alvarez-Roman et al. studied the entry and distribution of plastic particles throughout skin tissue, whereby fluorescent polystyrene particles with diameters of 20 and 200 nm were placed on skin tissues from a pig. They revealed that a higher amount of 20 nm polystyrene NPs was concentrated in the hair follicles in comparison with the 200 nm polystyrene NPs using a confocal laser scanning microscope. However, neither NPs penetrated the stratum corneum. In contrast, Campbell et al. provided evidence that 20-200 nm NPs can penetrate a depth of 2-3 μm into the top layers of the skin, and Vogt et al. suggested that transcutaneous NPs were absorbed by Langerhans cells.98–100 One facilitating factor for NPs to enter the skin is UV radiation, as UV rays can damage and weaken the skin barrier.101\n\nThe entry of NPs into keratinocytes may induce cytotoxic reactions, such as oxidative stress, trigger inflammatory factors, or even result in cell death, either by apoptosis or necrosis. A previous study suggested that polystyrene particles can interact with biological membranes, allowing the penetration of lipid bilayer membranes, alteration of cell membrane structure, and ultimately impeding normal cellular function.102\n\nFigure 1 depicts the sources of nanoplastic and their routes of exposure in human.\n\nOnce NPs enter the human body, they enter the circulatory system and are then distributed throughout the body. The concentration and interaction of NPs with their target molecules (e.g., proteins, phospholipids, and carbohydrates) depend greatly on the size, surface chemistry, and charge of the target elements. Furthermore, the high free surface energy of NPs allows their adsorption onto biomolecules when they cross complex biological fluids. The interaction between NPs and biomolecules leads to the formation of ‘biomolecular coronas,’ where NPs are encompassed by a collection of biomolecules. The formation of biomolecular coronas depends on the physicochemical properties of the NPs and their exposure time to biological components in the environment.80 These biomolecular coronas modify the biological traits of NPs, such as the enhanced rate of translocation, by altering the form of NPs to adapt to the surrounding biological environment. This is because the corona can lower the NP surface energy, thereby promoting dispersion. In some circumstances, it also promotes the interaction between NPs and cell surfaces, thereby increasing the toxicity potential of NPs. In contrast, some NPs lose their targeting abilities owing to corona formation.103\n\nThere are two types of coronas: ‘hard’ and ‘soft.’ ‘Hard’ coronas are a somewhat reversible but tightly bound layer of biomolecules formed through adsorption of biomolecules on bare nanoparticle surfaces immediately after contact. On the other hand, ‘soft’ corona consists of a more loosely bound interaction of biomolecules on the surfaces of NPs, and they are readily exchangeable if exposed with other nanoparticles with higher energy surfaces in the environment. Unlike ‘hard’ corona, any subsequent encounter of the nanoparticles and different biomolecules in a pristine environment may result only in partial displacement of the original ‘soft corona by the new biomolecules. Biomolecules that are not displaced would serve as a memory of the corona to the previous environment to which the nanoparticles are exposed. In general, the composition of the corona depends on the environment through which the nanoparticles pass. Furthermore, NPs can travel across every component within organisms because of their small sizes and potentially retain biomolecules in their corona from every environment they encounter. If the nanoparticles are retained in the corona for a long duration, the nanoparticle-corona complex will be recognized by the cell membrane receptor.80 The consequences of corona formation include the biodistribution of NPs and their potential pathophysiological effects within organisms.\n\nCellular uptake of NPs. There are several routes by which NPs can be taken up by the cells. The main route of NPs uptake is through endocytic pathways, where adhesive interactions between NPs and channel or transport proteins occur. These pathways include phagocytosis, micropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis pathways. Phagocytosis is a process in which particles (> 0.5 μm) are ingested by cells. After ingestion, the particles are enclosed in membrane-bound vesicles, also known as phagosomes, to allow them to be targeted by lysosomes for enzymatic degradation. The process is initiated when the particles bind to specialized receptors on cell membranes. After binding, polymerization occurs at the site of ingestion, leading to the formation of pseudopodal extensions, which are extensive deformations of the plasma membrane. Pseudopodia encompass particles in a cup-shaped structure. As the particles are completely internalized, depolymerization of actin filaments begins at the base of the phagocytic cup, allowing them to seal up into phagosomes. As phagosomes mature, they undergo a series of membrane fusion and fission events with endosomal compartments, eventually binding with lysosomes and forming microbicidal phagolysosomes.104\n\nMacropinocytosis is an endocytic pathway involving nonspecific uptake of extracellular materials, including soluble molecules. The process involves inward folding of cells due to the fusion of the cell surface with the basal membrane to form vesicular structures and macropinosomes. The initiation of this actin-dependent process depends on the stimulation of growth factors such as colony-stimulating factor 1 (CSF-1), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). Thereby, the polymerization of actin filaments increases at the cell membrane, allowing membrane ruffling to occur. Some lamellipodia-induced ruffles continue to fold inward and fuse with the basal membrane, forming macropinosomes. The maturation process of macropinosomes is cell type-dependent. For instance, the maturation process in macrophages occurs by shrinking macropinosomes, which eventually fuses with lysosomes for enzymatic degradation.105\n\nClathrin-mediated endocytosis is a type of vesicular transport that involves the use of clathrin, a triskelion-shaped scaffold protein. In this process, clathrin assembles and polymerizes to form a coat around the cytoplasmic region of the invaginated membrane. The coat serves as a reinforced mold and can dissociate rapidly after the vesicle splits from the membrane. However, this process can only occur in the presence of phosphatidylinositol-4,5-biphosphate (PIP2) and adaptor proteins. The growth of clathrin-coated pits requires the BAR (Bin/amphiphysin/Rvs) domain protein and reorganization of the actin network.106\n\nCaveolar-mediated endocytosis is clathrin-independent endocytosis, where it forms a bulb-shaped plasma membrane invagination called caveolae. The formation of caveolae depends on the presence of the integral membrane protein caveolins and peripheral membrane protein caving. During this process, caveolae bud off from the plasma membrane and form endosomes.107 It was shown that 44 nm polystyrene particles were mainly absorbed through clathrin-independent endocytosis into human colon fibroblasts and bovine oviductal epithelial cells.108 Dos Santos et al. studied the cellular absorption of 40 and 200 nm carboxylated polystyrene NPs in several tumor cell lines and deduced that NPs were absorbed into cells via active transport. This suggests that the uptake of NPs differs depending on the cell type.109 Still, the absorption of NPs into human cells is greatly dependent on their size and surface chemistry. Furthermore, another study demonstrated the absorption of NPs into J774A.1 macrophage cells through micropinocytosis, phagocytosis, and clathrin-mediated pathways, whereas in A549 cell lines, NPs enter via caveolae- and clathrin-mediated endocytic pathways.\n\nPrevious studies have shown that the interaction between plastic particles and human cells is influenced by their high specific surface areas, charges, structures, and types of targeted cells.110\n\n\nNeurotoxicity\n\nRecent laboratory and field studies on aquatic organisms and other commercial species have suggested that NPs are mainly absorbed in the GI tract and are distributed to other parts of the body. Once NPs enter the blood circulation, their nanosize facilitates translocation across biological barriers, and ultimately across the blood-brain barrier (BBB). Ding et al. revealed that MPs could enter the brain of Oreochromis niloticus through the circulatory system.111 Geiser et al. further supported the hypothesis that NPs are present in the brain of fish through the ingestion of water or food across the BBB in an in vitro study.112 Mattsson et al. showed that both 53 and 180 nm sized polystyrene plastic particles can enter the brain of crucian carp (Carassius carassius).113 Studies have shown that nanoparticles can reach the brain directly via nerve fibers in the olfactory epithelium (nervus olfactorius) located at the roof of the nose. This method has been demonstrated using transsynaptic transport. Although this method allows nanoparticles to bypass the blood-brain barrier, its occurrence is extremely rare.114\n\nAs it has been proven that NPs can cross the BBB, it is essential to evaluate the toxic effects of NPs on brain tissue. Several studies have proposed that NPs can accumulate in these tissues, exerting multiple adverse effects on the nervous system. In this context, the toxic effects of NPs on the brain depend on the exposure duration, size, and type of NPs, as well as indirect mechanisms, such as oxidative stress.115 Wan et al.116 revealed that neurotoxicity caused by exposure to polystyrene MPs in zebrafish larvae may be associated with some metabolic changes. Ding et al. demonstrated decreased levels of amino acids phenylalanine and tyrosine in adult tilapia after exposure to polystyrene NPs. These amino acids play a significant role in neurological function. Ding et al. concluded that NPs can interact with these amino acids, which eventually alters their metabolic pathways, and thus alters the formation of various neurotransmitters.115\n\nFurthermore, NPs can induce oxidative stress-related damage in the brain. It has been reported that 1 to100 μm sized particles exert more severe stress than 0.3 μm polystyrene MPs, which indicates that larger particles, though in the micro range, can be more harmful. This may be explained by the hypothesis that medium-sized MPs can travel more freely across cells while inducing mechanical injuries and alterations in biochemical pathways, including oxidative damage and inflammatory responses.115 The brain is extremely sensitive to oxidative stress due to several factors. Oxidative stress is a biological condition in which redox homeostasis, which is an equilibrium between oxidant and antioxidant levels maintained in vivo, fails to occur. The imbalance in this equilibrium between free radical production and cellular antioxidants can be due to several disorders and/or diseases, such as carcinogenesis, inflammation, and aging. Chen et al. developed methods to evaluate the role of oxidative stress in neurotoxicity via specific biomarkers of oxidative stress, such as catalase (CAT), glutathione, peroxidase activity, and reduced glutathione.117,118\n\n\nSensitivity of the brain to oxidative stress\n\nFirst, the brain is one of the most metabolically active organs and consumes an approximate amount of 20-30% of inspired oxygen, despite it only accounting for 2% of the body weight (approximately 1300 g).119 The need for high oxygen levels in the brain is because of the substantial amount of adenosine triphosphate (ATP) required in ion channels for activities such as action potentials and neurosecretion to maintain neuronal intracellular ion homeostasis.120\n\nSecond, most areas of the brain (e.g., substantia nigra, caudate nucleus, putamen, and globus pallidus) are rich in iron. Iron plays a key role in oxygen transport. For instance, the redox cycle between ferrous and ferric ions is essential for various electron transfer reactions, which are important for several cellular processes, including neurotransmitter synthesis, myelination of neurons, and mitochondrial function.121 Hence, defective iron homeostasis can result in the production of neurotoxic reactive species (e.g., such as reactive oxygen species (ROS)), eventually leading to oxidative damage and cell death in the central nervous system. Because ROS are highly unstable, they can easily react with a majority of macromolecules, such as proteins, nucleic acids, and lipids.121 Moreover, this reaction is further exacerbated, especially in the brain, because neuronal cells are unable to neutralize free radicals.119 Several studies have demonstrated that NPs can cause ROS accumulation.\n\nAdditionally, neuronal membranes are rich in polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA).120 PUFAs are especially vulnerable to oxidative stress because of their unsaturated double bonds. Recent discoveries have revealed that ROS can cause lipid peroxidation (LPO), a process that involves the disruption of oxidants on lipids that consist of C=C double bonds, such as PUFAs. In this context, ROS attack cellular membrane PUFAs and cause functional and/or structural impairments. Consequently, a group of highly reactive α, β-unsaturated aldehydes (e.g., 4-hydroxy-2-noneal (HNE), malondialdehyde (MDA), and acrolein) were generated.121 These highly reactive aldehydes are highly diffusive, allowing them to attack or generate covalent linkages with other cellular constituents, eventually leading to extensive cellular damage.\n\nThere are three types of mechanisms that result in LPO production-free radical-mediated oxidation: free radical-independent, nonenzymatic oxidation, and enzymatic oxidation.120 In the case of NP exposure, it is presumed that LPO occurs following free-radical-mediated oxidation. This mechanism involves a cascade of processes in the oxidative deterioration of lipids, such as initiation, propagation, and termination.121 In the last step, termination of propagation steps in LPO can only be performed with the complete utilization of the substrates or through interaction with antioxidants such as vitamin E and C.121 Otherwise, these cyclized fatty acids will only proliferate further while damaging the components of the cellular membrane, such as lipids and proteins, and further generate by-products of LPO.\n\nIn general, LPO affects several functions of the central nervous system (CNS) through membrane rigidity, decreased enzymatic activity in the membrane, damage to membrane receptors, and altered permeability of biological membranes.121 Consequently, these reactions lead to altered cellular function and severe cytotoxicity, resulting in uncontrolled cellular growth or apoptosis. In the CNS, PUFAs disrupt neuronal homeostasis, eventually causing brain dysfunction. After NP exposure, Barboza et al.122 reported higher levels of LPO and acetylcholinesterase (AChE) activity in the brains of Dicentrachus labrax, Trachurus, and Scomber colias. The increased level of AChE activity can be explained by the higher levels of LPO damaging acetylcholine-containing vesicles, thus causing a rise in neurotransmitter levels released in the synaptic clefts.\n\nAChE is a cholinergic enzyme that is mainly produced at the postsynaptic neuromuscular junctions. AChE breaks down the neurotransmitter acetylcholine (ACh) into acetic acid and choline to ensure the termination of cholinergic neurotransmission in neuromuscular junctions and cholinergic brain synapses.123 In general, AChE is important for the regulation of brain function and is thus considered a biomarker for neurotoxicity. A study on zebrafish revealed that NP exposure can decrease AChE levels and inhibit neurotransmitters, such as dopamine, melatonin, aminobutyric acid, and serotonin in zebrafish.124 This is further supported by other studies on Medaka, O. latipes, and Pormatoschistus microps after exposure to MPs and NPs.\n\nInhibition of AChE activity can lead to several adverse effects, such as paralysis and, in severe cases, death.123 Furthermore, inhibition of AChE activity can be determined by developmental and motility alterations. For instance, the exposure of zebrafish to NPs suppresses locomotor ability, which leads to the deduction that decreased AChE activity is due to the binding of important channels, thus preventing AChE production. In addition, most studies have scrutinized the exposure of MPs and NPs to neurotoxicity through the evaluation of behavioral anomalies.125 Uncontrolled movements after exposure to NPs are inferred by the altered expression of essential neurotransmitters, such as ACh, glutamate, and γ-aminobutyric acid, or defective activation of neurons.\n\nRecent discoveries have been made regarding the association between gene deregulation and exposure to NPs. For instance, the expression of glial fibrillary acidic protein (GFAP) and alpha-1 tubulin (α1-tubulin) in the CNS during the initial stages of zebrafish development is an important biomarker of neurotoxicity. In this context, α1-tubulin is essential for the formation of microtubules, whereas GFAP is important for astrocyte development. According to the US Environmental Protection Agency (EPA), upregulation of GFAP indicates neurotoxicity.124\n\n\nNP neurotoxicity\n\nTo date, there have been a very limited number of studies on the neurotoxic potential of NPs in vitro. In vitro studies have largely focused on the extent of NP absorption and its implications on cells, such as proinflammatory effects and genotoxicity, based on particle size. Furthermore, cell models allow the study of NP-induced neurotoxic mechanisms, including oxidative stress, routes of NPs integrating into mitochondria, immune responses after exposure to NPs, interaction between NPs and enzymes, and alteration of receptor or channel function of neuronal cells. Generally, in vitro studies have revealed the many effects of NPs on fundamental cellular functions, including proliferation, differentiation, and apoptosis, to determine their neurotoxicity.126 However, the mechanism of neurotoxicity greatly depends on the surface area, composition, type, and shape of NPs, as well as the absorptivity of NPs into the body.\n\nHuman cell lines. A 3-dimensional (3D) in vitro model of a human embryonic stem cell (hESC)-derived (WA09) was used to test the potential developmental neurotoxicity exerted by NPs because of its ability to imitate conditions in vivo.126 The WA09 cells were differentiated into PAX6+ neural precursor cells. Methylmercury, a developmental neurotoxicant, was used to test the sensitivity of the 3D model in detecting developmental neurotoxicity (DNT). After exposure to methylmercury, a significant decrease in the expression of neuronal precursor and neuronal marker genes indicated that the 3D model reacted sensitively with methylmercury.\n\nPolyethylene nanoplastics (PE-NPs) (30 nm, anisotropic nanocrystals, ζ potential -30mV) were used because of their ability to remain inert under physiological conditions, including retaining non-aggregation in the culture medium for a prolonged period (>3 months).126 This allows for the investigation of the biological effects of bio-persistent NPs upon chronic exposure. The acute and chronic toxicities exerted by the fluorophore-incorporated PE-NPs were recorded using flow cytometry. Acute toxicity was measured after 48 h in HeLa cells, with an EC50 value of 450 μg/ml, in which the ATP content decreased with increasing concentrations of PE-NPs.\n\nIn addition, LUHMES (human neuronal precursors) cells, a model closer to neural tissue, were used to incubate PE-NPs instead of cervical tissue-derived HeLa cells. Similarly, LUHMES cells were incubated for 48 h and an EC50 value of 257 μg/ml was determined. As the concentration of PE-NPs increased, the ATP content decreased. However, unlike in HeLa cells, the initial decrease in ATP content was constant but minimal. A significant decrease in ATP content was observed only when the PE-NPs reached a concentration of 90 μg/ml, which is likely indicative of the cellular oxidative stress exerted by PE-NPs. These findings strongly suggest that PE-NPs can initiate stress in mitochondria by reducing ATP content in a dose-dependent manner.126\n\nBefore chronic toxicity was measured, the incorporation of PE-NPs into neurospheres was also assessed. Fluorophore-labelled PE-NPs were incorporated into neurospheres at increasing concentrations and the results were recorded after 24 and 48 h. Unsurprisingly, PE-NPs were clearly recorded inside the neurospheres at a concentration of 360 μg/ml at the 48-hour mark. To confirm this, flow cytometric analysis of single-cell suspensions showed that PE-NPs could penetrate and incorporate into the neurospheres, as most of the cells (84 ± 6%) were positive at the highest concentration used (1440 μg/ml). An ATP concentration of 696 μg/ml (EC50) and decreasing ATP content with increasing PE-NP concentration were noted. Together with increased malondialdehyde concentrations, these findings may indicate increased oxidative stress levels in the cells and highlight the ability of PE-NPs to pass through the structural barrier into the cells without using altered forms.126\n\nThe chronic toxicity of PE-NPs was assessed after 18 days using cell viability assessment. EC50 values of 296 and 191 μg/ml were recorded using the resazurin reduction and ATP content as parameters, respectively. For both parameters, a similar trend of decreasing cell viability with increasing PE-NP concentration was observed. Another study on the differentiation and maturation process of neural precursor cells was conducted by exposing neurospheres to 22.5 μg/ml PE-NPs (a non-cytotoxic concentration). The results showed that PE-NPs interfered with the downstream expression of NOTCH pathway genes (HES5 and NOTCH1), NEUROD1, ASCL1, and FOXG1. PAX6 was not affected, suggesting that PE-NPs only affected later neuronal progenitors.126 In summary, this study revealed that PE-NPs caused severe impairment of the nervous system (DNT) due to dysregulated gene expression. Remarkably, all affected genes play a vital role in neurodevelopment; for instance, the NOTCH pathway ensures proper embryonic neural development and adult brain plasticity.127 Additionally, ASCL1 regulates neurogenesis by deleting defects in the ventral telencephalon,128 and FOXG1 is responsible for repressing specific transcription during early telencephalon development. PE-NPs can affect progenitor cells and neuronal development by affecting the genes responsible for neurogenesis. Furthermore, increasing concentrations of PE-NPs may cause cytotoxicity in neural cells, thereby manifesting as DNT.126\n\nAnother study used varying concentrations of NPs to determine their direct effect on cells in the nervous system. In this study, polystyrene NPs (50 nm, negatively discharged sulfonated surface, spherical) were added to human neuroblastoma cells SH-SY5Y (seeding density of 1.3×104 cells/cm2, cultured for 82 h), and the effects were compared to those of acrylamide, a potent neurotoxin (polystyrene NP concentration: 2, 10, and 50 μl/ml; acrylamide concentration: 0.8, 20, and 500 μg/ml). The viability of cells in polystyrene NP-containing medium at concentrations of 10 and 50 μl/ml was lower than that at 2 μl/ml. The morphology of cells in the polystyrene NP-containing medium differed from that in the acrylamide-containing medium with the appearance of decreasing processes, fuzzy cell bodies, and more granular substances (a few μm in size) surrounding the cells. Furthermore, the granular substances consisted of nucleic acid fragments, indicating necrosis, where the integrity of the organelle and plasma membrane had been breached, leading to the release of cytosolic and intracellular components into the surrounding environment. Thus, this study demonstrated the induction of cytotoxicity in SH-SY5Y cells when exposed to polystyrene NPs, whose toxicity might be on par with or exceed that of acrylamide. Morphological alterations, swelling of nuclei, release of organelle components, and shrinkage of neurite outgrowth deduced the neurotoxicity of NPs in vitro.129 This study also provides insight into the concentration of polystyrene NPs required to induce cytotoxicity. Based on these results, higher concentrations caused more apparent adverse effects on the neuronal cells. However, the dose or amount required to trigger cytotoxicity based on experimental models may differ from the threshold of the human brain. This is because NPs must overcome several obstacles, including the blood brain barrier (BBB), glial cells, and phagocytes of the central nervous system. Hence, the concentration of NPs used in this study may not reflect the manifestation in the actual human brain tissue.\n\nRodent cell lines. A study investigated neurotoxicity of NPs using different concentrations of polystyrene (PS) NPs (nanoscale-PS latex beads, 100 nm diameter, ζ potential -40.60 ± 3.96 mV) towards three different primary cell types from CD-1 (ICR) mice, including mouse embryonic fibroblasts (MEFs), mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes.130 The experiment was conducted on the cell types by measuring two parameters, including cellular viability and level of the apoptosis marker cleaved caspase-3 (CC3). To test cellular viability, cells were incubated with different concentrations of PS-NPs (0, 50, 100, and 200 mg/L) for two days and the MTT assay was performed. The results revealed that MEFs and astrocytes were not affected, with no change in viability at concentrations of 200 and 100 mg/L, respectively. However, the viability of mixed neuronal cells significantly decreased at a concentration of 100 mg/L. Apoptosis markers, analyzed using immunofluorescence, revealed that CC3 increased upon exposure to PS-NPs at 100 mg/L, with higher levels in mixed neuronal cells after 2 days, but there was no change in CC3 levels in MEFs and astrocytes. However, the levels of lipocalin-2 (Lcn2) and proinflammatory cytokines, rarely detected under standard culture conditions, were elevated upon exposure to PS-NPs. This indicated the induction of reactive astrocytosis, but astrocytes did not undergo apoptosis.\n\nThe study also revealed that increasing concentrations of PS-NPs and plasma membrane permeability greatly enhanced the deposition and accumulation of PS-NPs, which in turn reduced cellular viability and increased apoptosis observed from the neuronal marker gene expression, Tubb3, and glial marker, GFAP. Tubb3 decreased, whereas GFAP increased at a concentration 100 mg/L of PS-NPs in mixed neuronal cells, suggesting that exposure to NPs leads to defective neuronal development, neuronal death, and accumulation of neuronal cell bodies. In astrocytes, GFAP decreased after exposure to PS-NPs, whereas the proinflammatory cytokines TNFa and IL-1b increased, indicating reactive astrocytosis.130 To summarize, this study revealed the toxicity of PS-NPs on neuronal cells through apoptosis. Furthermore, this investigation provides the insight that NPs neurotoxicity is dependent on cell type, as evident from the difference in PS-NP deposition and accumulation in different cells.\n\nIn contrast to in vitro studies, the neurotoxic potential of NPs has been extensively investigated in animal models. In vivo studies have highlighted the entry of NPs into experimental modules via multiple routes, including dietary, respiratory, or epidermal pathways. The behavioral changes in the experimental animals were also emphasized, providing more comprehensive data on the absorption, distribution, metabolism, and excretion of NPs in the body.\n\nRodents. One study in rodents investigated the neurotoxicity of pristine polystyrene NPs (25 and 50 nm) on neurobehavioral effects in animals. Four different dosages (1, 3, 6, and 10 mg/kg of body weight) of NPs were administered to four groups of adult Wistar male rats (6-8 weeks old; 190 ± 10 g) for 35 days through oral gavage using a 5 ml gavage needle to the pharyngeal region to avoid particle loss. At the end of the final week, several neurobehavioral tests were performed on six consecutive days to assess behavioral domains, including total locomotor activity, spatial working memory, anxiety-related behavior, motor coordination, and passive avoidance memory performance.\n\nThe results from the different tests were mixed: total locomotor activity in the open field, Y-maze test to assess spatial working memory, and passive avoidance test to investigate memory retention were normal for each rat in the four treatment groups. The elevated plus maze test to analyze anxiety-like behaviors in the rats was also normal, except for rats that received 10 mg polystyrene NPs/kg of body weight/day, which showed greater open arm entries, indicating reduced anxiety. The coordination and balance of rats receiving 3 mg polystyrene NP/kg of body weight/day, as assessed by the rotarod test, decreased compared to baseline values.131 Overall, this study suggests that pristine polystyrene NPs did not cause any significant adverse effects on the behavior of adult rats.\n\nInvertebrates. In one neurotoxicity study, Caenorhabditis elegans was exposed to different concentrations of PS-NPs (25, 50, and 100 nm) for 72 h.132 This study highlighted the effect of PS-NPs on the growth and locomotion of C. elegans, induction of dopaminergic loss and mitochondrial damage, as well as oxidative stress and apoptosis. To assess growth and locomotion, C. elegans was exposed to different concentrations (10 and 100 μg/L) for 72 h, and parameters such as body length, head thrashes, and body bending were recorded. The results revealed that increasing concentrations of PS-NPs stunted body length, and the effect was more significant for larger particles (50 and 100 nm). In addition, 50 and 100 nm PS-NPs exerted obvious locomotor deficits.\n\nIn terms of head thrashes and body bends, the results revealed a reduction of 17.2% and 28.3% at 10 μg/L 50 nm PS-NP exposure and 29.0% and 37.0% at 100 μg/L 50 nm PS-NPs, respectively. As for 100 nm PS-NPs at the concentrations of 10 and 100 μg/L, head thrashes reduced by 17.4% and 29.2%, and body bends by 20.3% and 36.3%, respectively. These results indicated that PS-NPs exerted neurotoxicity by affecting C. elegans locomotion, and the effects were influenced by the size and concentration of PS-NPs.132\n\nTo further assess the neurotoxicity of PS-NPs, induction of dopaminergic loss and mitochondrial damage in C. elegans was evaluated. In this study, BZ555 strain transgenic C. elegans was exposed to a range of PS-NP concentrations, and fluorescently labelled dopaminergic neurons were measured. Researchers found that only 100 μg/L showed downregulation of dopaminergic content by 8.2%, 11.3%, and 12.2% at 25, 50, and 100 nm NPs, respectively. Furthermore, mitochondrial dysfunction was observed by labelling the mitochondria with green fluorescent proteins in the transgenic PD4251 strain C. elegans. A reduced fluorescence signal was noted after exposure to PS-NPs, and 100 nm particles revealed a lower fluorescence intensity at 10 and 100 μg/L when compared with those at 25 and 50 nm. Mitochondrial damage affects the mechanisms required for neurodevelopment, implying that PE-NPs exert neurotoxicity.\n\nFinally, previous study evaluated oxidative stress and apoptosis in C. elegans following exposure to PS-NPs. Mitochondria play a significant role in ensuring a delicate balance of reactive oxygen species (ROS) levels, and excess production of ROS results in several disorders. In this test, oxidative stress induced by PS-NPs was measured using the DCFH-DA assay. Although there was no change in C. elegans exposed to 10 μg/L of 25 nm PS-NPs, an increased intracellular ROS level was noted in 100 μg/L of 25 nm PS-NPs was noted. Furthermore, both 50 and 100 nm NPs at concentrations of 10 and 100 μg/L showed a significant increase in ROS levels, with 100 nm PS-NPs recording the highest value. Apoptosis levels were measured based on lipofuscin and lipid peroxidation levels (oxidative stress markers). The results showed that higher concentrations of larger PS-NPs caused increased lipofuscin levels, which indicated a higher rate of apoptosis.132 In conclusion, PS-NPs exerted significant neurotoxicity in C. elegans depending on their concentration, and the extent of neurotoxicity was inversely proportional to the size of the NPs.\n\nAnother study was conducted on exposure to PS-NPs and its neurotoxicity in nauplii of brine shrimp Artemia fransiscana. To further understand the neurotoxicity of PS-NPs, A. fransiscana was considered a suitable model for planktonic species. In this study, 50 nm cationic amino-modified PS-NH2 was used at concentrations of 0.1, 1, 3, and 10 μg/mL, and each concentration was tested by incubating in a short- (48 h) and long-term exposure duration (14 days). The neurotoxic potential of PS-NPs was determined in accordance with physiological, chemical, and molecular responses. Physiological endpoints include growth, development, and feeding behaviors, whereas at the molecular level, the parameters are processes such as biotransformation, neuronal transmission, oxidative stress, and stress response. All endpoints were evaluated based on the activity of carboxylesterases (CbE), cholinesterase (ChE), glutathione-S-transferase (GST), CAT, LPO, and heat shock protein.\n\nResults demonstrated that the highest mortality of over 70% was observed at highest concentration (10 μg/mL) and short-term exposure (48 h) evaluated using enzymatic activities. In this context, CbE was significantly inhibited, whereas ChE was unaffected in a concentration-dependent manner. There was a significant reduction in total body length at 1 and 10 μg/mL PS-NPs, but no reduction was observed upon exposure of 0.1 μg/mL PS-NPs. In comparison with long-term exposure (14 days), mortality rates were significantly different with increasing concentrations of PS-NPs. Developmental alterations were observed after 3 days of exposure to 1 and 3 μg/mL PS-NPs. Feeding behaviours, evaluated as the rate of ingestion and filtration, did not change after exposure to 0.1 and 1 μg/mL PS-NPs for 14 days. As for the oxidative response and detoxification, both GST and CAT levels were significantly decreased upon exposure. No differences were observed in GST between 0.1 and 1 μg/mL, and only CAT levels were significantly affected by exposure to 1 μg/mL.133\n\nIn summary, short-term exposure to PS-NPs may affect the growth and development of the brine shrimp Artemia fransiscana in a concentration-dependent manner, with an increase in oxidative stress. After long-term exposure to NPs, the survival of the test models was impaired due to neurotoxic effects. The presence of LPO, together with decreased ChE activity, indicates an accumulative effect.\n\nTable 1 summarizes the neurotoxicity of NPs in different models.\n\n\n\n- Acute toxicity: 2 days (48 hours)\n\n- Chronic toxicity: 18 days\n\n\n\n- Acute toxicity:\n\n• Decrease ATP content in both HeLa and LUHMES cells. (Depends on concentration of PE-NPs)\n\n• Increase concentration PE-NPs, decrease ATP content.\n\n- Penetration of PE-NPs into cells:\n\n• Concentration of fluorophore-labelled PE-NPs were easily observed in cells after 48h with increasing concentration of PE-NPs exposure.\n\n- Chronic toxicity:\n\n• Cell viability decreased (observed with increase resazurin reduction and ATP content).\n\n• NOTCH1, HES5, NEUROD1, ASCL1, FOXG1 downregulated, PAX6+ no changes.\n\n\n\n- Distorted appearance and reduced cell viability observed at concentration of 10 and 50 μl/ml PS-NPs.\n\n- Observation including:\n\n• Decreasing processes\n\n• Fuzziness of cell bodies\n\n• A lot of granular substances (a few μm in size) was seen to surround the cells (consist of nuclei acid fragments)\n\n• Swelling of nuclei\n\n• Shrinkage of neurite outgrowth\n\n\n\n- Mouse embryonic fibroblasts (MEFs)\n\n- Mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes\n\n\n\n- Cellular viability:\n\n• Significant reduction of mixed neuronal cells at concentration 100 mg/L, not affected by others at 200 mg/L.\n\n- Apoptosis level CC3:\n\n• Increased in mixed neuronal cells, not affected by others at 200 mg/L.\n\n• Lcn2 and proinflammatory cytokines increased in astrocytes.\n\n- Gene expression:\n\n• Reduced neuronal marker Tubb3 and GFAP in mixed neuronal cells at 100 mg/L.\n\n• Reduced GFAP, increased Tnfa and IL-1b in astrocytes.\n\n\n\n- No significant results were observed.\n\n\n\n- Growth and locomotion\n\n• Increase concentration PS-NPs decreased body length of C. elegans. (Growth)\n\n• Smaller size C. elegans are more affected. (Growth)\n\n• Reduced head thrashes and body bends at higher concentration and smaller sizes of PS-NPs. (Locomotion)\n\n- Dopaminergic loss and mitochondrial damage\n\n• Only 100 μg/L shows result where dopaminergic content was downregulated. Larger size PS-NPs cause greater effect.\n\n• Reduced fluorescence signal exerted by labelled mitochondria, more affected at larger sizes and higher concentration.\n\n- Oxidative stress and apoptosis\n\n• Increase intracellular ROS level in all sizes and concentration, with 100nm at 100 μg/L showed highest.\n\n\n\n- Oxidative stress\n\n• Decrease in antioxidant enzyme\n\n- Occurrence of LPO\n\n• Decrease activity of cholinesterase\n\n- Lower survival rate under long term exposure\n\nTo understand the association of NPs with neurodegenerative diseases, it is important to understand the interaction of NPs with proteins. As mentioned previously, the internalization of NPs allows interaction with biomolecules in the cytoplasm (in particular, proteins). The binding mechanism between proteins and NPs mainly involves weak interactions, including hydrophobic interactions, hydrogen bonds, van der Waals attraction forces, and electrostatic forces. The interaction of NPs with proteins can result in three consequences: (1) protein corona formation, (2) protein-induced coalescence of NPs, and (3) conformational changes in the protein secondary structure.134 Formation of a protein corona allows NPs to escape immune system surveillance and further impede the functions of cellular and molecular processes.134 Furthermore, the presence of a protein corona allows NPs to interact with each other through protein-protein interactions, thereby promoting coalescence and aggregation.135\n\nThe effects of NPs on the secondary structure of proteins are critical. This is because the function of a protein is dependent on its three-dimensional structure, and a slight alteration in protein structure may lead to protein misfolding and functional loss. Holloczki et al.136 demonstrated that NPs can modify the protein structure and ultimately lead to protein misfolding. The interaction between NPs and proteins is dependent on the presence of apolar side chains of amino acids, such as phenylalanine and tryptophan, which are more susceptible to adsorption on the surface of NPs due to hydrophobic interactions. Furthermore, the team investigated the interactions between NPs and small peptides with different secondary structures (such as α-helices and β-sheets). In this context, it was demonstrated that polyethylene NPs promote α-helix formation, whereas nylon NPs increase the presence of β-sheets and unfold helical domains. This indicates that NPs can result in protein misfolding.135\n\nThere is little evidence to imply that NPs can induce neurodegenerative diseases. However, studies related to nanomaterials have been conducted to study the potential risks that nanoscale objects can introduce in neurodegenerative diseases. Most nanoparticles can easily access bodily compartments, including the brain. An in vitro study conducted to understand how nanoparticles promote the formation of amyloid fibrils (by assembling proteins through nucleation mechanisms) showed that nanoparticles are encompassed by proteins once they are in contact with biological fluids and protein coronas are formed. This state can either denature the protein or remain unchanged, depending on the surface charge, hydrophobicity, and intrinsic stability of the proteins and nanoparticles.137\n\nThe binding of proteins to nanoparticles with large surface-to-volume ratios can eventually increase the chances of partial unfolding of proteins. As the levels of these abnormal proteins increase, so do the chances of association with one another, and this process may form new radical protein clusters. Furthermore, nanoparticles can affect protein self-assembly reactions, leading to the disruption of certain essential biological processes and eventually inducing protein misfolding-associated diseases. Amyloid fibrils are formed through the assembly and aggregation of misfolded proteins, forming insoluble fibers that are resistant to degradation. Neurodegenerative diseases accompanying amyloid fibril formation include Alzheimer’s disease (AD) and Creutzfeldt-Jacob disease.137\n\nAD is a neurodegenerative disorder characterized by impaired cognition and behavior that can significantly affect a patient’s daily function. AD is initiated by the accumulation of senile plaques (SPs), also referred to as beta-amyloid plaques. SPs accumulate before the clinical onset of AD, and this is followed by a loss of cognitive function when neurofibrillary tangles (NFTs) are formed and eventually the loss of neurons. SPs mainly consist of insoluble deposits of proteins and cellular components surrounding neurons. This plaque is constructed of beta-amyloid proteins snipped from a larger protein, namely, the amyloid precursor protein. The fragments subsequently aggregated to form plaques.\n\nEventually, plaques are deposited in the hippocampus and in other areas of the cerebral cortex. In a healthy person, the microtubules in their neurons are stabilized by a type of protein, tau, to ensure proper transportation of nutrients from the cell body all the way to the end of the axon, and vice versa. However, tau proteins in patients with AD are chemically altered; they are tangled with each other and accumulate in neurons. Eventually, distorted tau proteins cannot bind to microtubules, resulting in disintegration and impaired communication between neurons, further causing neuronal death and thus forming NFTs. It is still unknown if the formation of SPs is a by-product of AD or if the plaques are the initial trigger for AD. However, beta-amyloid fibrillar proteins are extremely neurotoxic to neurons. Furthermore, the fibrillar form of beta-amyloid proteins has been shown to affect the phosphorylation state of the tau protein.\n\nAnother speculation is that NFTs lead to the formation of SPs, as the absence of normal tau proteins destabilizes microtubular systems, eventually distorting the function of the Golgi apparatus, forming abnormal proteins, and increasing amyloid-beta protein production. NPs could be a risk factor for inducing the formation of NFTs or SPs because of their ability to enhance the probability of protein misfolding and promote the self-assembly of proteins into amyloid fibrils.138\n\nLiang et al. suggested that NPs can potentially trigger Parkinson’s disease (PD)-like neurodegeneration in mice based on brain single-nucleus transcriptomics. Several approaches were applied to understand the potential neurotoxicity of NPs by exposing 50 nm polystyrene NPs at doses ranging from 0.25-250 mg/kg on mice orally for 28 days. The evaluation methods include neurobehavior, magnetic resonance imaging (MRI), neuropathological examination, and transcriptomics. Moreover, transcriptional changes were observed in the mouse brain using single-nucleus RNA sequencing (snRNA-seq) techniques, providing insights into the transcriptomic analysis of cell populations at the single-cell level. This method was further confirmed by evaluating the levels of misfolded proteins, inflammation, and adenosine triphosphate (ATP) in different areas of the mouse brain (midbrain, hippocampus, striatum, and cortex).139\n\nFirst, researchers investigated the ability of polystyrene NPs to penetrate the BBB and enter the mouse brain tissue through Evans blue staining. The intensity of staining was found to increase with increasing concentrations of polystyrene NPs, and these were dispersed throughout the brain, including the cortex, hippocampus, substantia nigra pars compacta (SNc), and striata.\n\nTo evaluate the effect of polystyrene NP on neurobehavior, such as the spontaneous activity of mice, locomotion, and anxiety levels, the rotarod test (for coordination and balance), grip strength, and distance travelled in an open field were assessed. The results demonstrated that the mice exhibited shorter travelled distances, weakened grip strength, and increased chances of falling off the rod after exposure to increasing concentrations of polystyrene NPs. Although neurobehavioral deficits were detected, symptoms of PD such as resting tremors and muscle rigidity were not observed. Furthermore, studies have revealed that Nissl bodies and dopaminergic neurons are lost in the SNc and the striatum.\n\nAn snRNA-seq test revealed that polystyrene NPs can induce cell-specific responses, with the responses mainly linked to disorders in energy metabolism and failure in mitochondrial function, especially in excitatory neurons. This test was further verified by the decreased ATP content and expression levels of ATP-associated genes and proteins. Inflammatory instability can be observed in astrocytes and microglia, with further dysregulation of synaptic functions and proteins in astrocytes, oligodendrocytes, and endotheliocytes.\n\nAll the above-mentioned complications are indications of PD-like neurodegeneration, especially with the induction of diminished motor function. Implications such as decreased Nissl bodies, dopaminergic neurons, or level of ATP content mainly occur in the SNc and striatum, which are the main lesion sites of PD. Dopaminergic neurons could not be distinguished by snRNA-seq, but inhibition of the ATP metabolic gene and mitochondrial dysfunction were mainly manifested in excitatory neurons, including dopaminergic neurons. This could be a major cause of dopaminergic neuron depletion, thereby promoting PD-like neurodegeneration.139\n\n\nConclusion\n\nThe use of plastics is inevitable in daily life. The growing production and usage of plastics have contributed to the accumulation of plastic waste. Unfortunately, many levels of society are still oblivious to proper management of plastic waste, resulting in environmental pollution. Plastic waste can be degraded into smaller fragments, such as MPs, and further into NPs if exposed to factors such as UV radiation, weathering, and biodegradation under prolonged exposure.\n\nHumans are then exposed to MPs and NPs, which are accessible because of their minute dimensions, enabling them to travel to any anatomical region without further resistance. Scientists have proven the possibility of MPs and NPs entering the human body via the ingestion of food or drinking water, inhalation, or contact. Moreover, several articles have also highlighted the various toxicities that MPs and NPs can induce in humans, in particular neurotoxicity. Compared to MPs, NPs have become intensely investigated in emerging research due to their enhanced capacity to move freely across biological components and increased toxicity due to their nano-characteristics.\n\nMost concerns on NP toxicity lean towards the central nervous system, and NPs are regarded as a worse ‘extension’ of MPs in many circumstances. Evidence shows that the ability of NPs to pass through the BBB into the brain tissue may lead to neurotoxic mechanisms, such as induction of oxidative stress and inhibition of AChE activity in vitro and alteration of neurotransmitter levels or behavioral changes in vivo. However, clinical studies of human exposure are lacking.\n\nMany questions remain to be answered in the quest to understand the impact of NPs on the human body. Most research has been conducted on cell lines, which overlook physiological processes, such as the body’s immune reaction to the entry of foreign substances. Detection methods or instruments to accurately measure the concentration of NPs in the environment and within the body are yet to be defined, as the techniques are predominantly experimental. Even the definition of ‘NPs’ are not well understood, as a relatively novel research field. While NPs are evidently present in the aquatic environment, data are scarce on the threshold levels that will impact the aquatic system and food chain, causing bioaccumulation and potential risks to humans.\n\nEvidence of NP neurotoxicity is even less explicit, and most studies have depended on in vitro experiments. Future work should strive to elucidate the effect of leached chemicals and plastic materials, the concentration of NPs in the environment, chronic exposure, and risk assessment of NPs in humans. The focus should also include NPs from terrestrial environments in addition to marine environments.\n\n\nEthics and consent\n\nEthical approval and consent were not required.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nWe thank the IMU University for their support of this project.\n\n\nReferences\n\nThompson RC, Swan SH, Moore CJ, et al.: Our plastic age.[cited 2022 Nov 26]. Reference Source\n\nAndrady AL, Neal MA: Applications and societal benefits of plastics. Philos. Trans. R. Soc. B Biol. Sci. 2009 Jul 7 [cited 2022 Nov 26]; 364(1526): 1977–1984. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeeker JD, Sathyanarayana S, Swan SH: Phthalates and other additives in plastics: human exposure and associated health outcomes. Philos. Trans. R. Soc. B Biol. Sci. 2009 Jul 27 [cited 2022 Nov 26]; 364(1526): 2097–2113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe C ompelling Facts About Plastics Ananalys is of plastics producti on, demand and recover y for 2006 in Europe.Published in January 2008.\n\nChamas A, Moon H, Zheng J, et al.: Degradation Rates of Plastics in the Environment. ACS Sustain. Chem. Eng. 2020 Mar 9 [cited 2022 Nov 26]; 8(9): 3494–3511. Publisher Full Text\n\nPlastic production worldwide 2020\|Statista: [cited 2022 Nov 26]. Reference Source\n\nPlastic Pollution - Our World in Data: [cited 2022 Nov 26]. Reference Source\n\nPlastics and the Environment - Google Books: [cited 2022 Nov 26]. Reference Source\n\nBarnes DKA, Galgani F, Thompson RC, et al.: Accumulation and fragmentation of plastic debris in global environments. Philos. Trans. R. Soc. B Biol. Sci. 2009 Jul 27 [cited 2022 Nov 26]; 364(1526): 1985–1998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTopçu EN, Tonay AM, Dede A, et al.: Origin and abundance of marine litter along sandy beaches of the Turkish Western Black Sea Coast. Mar. Environ. Res. 2013 Apr 1; 85: 21–28. PubMed Abstract \| Publisher Full Text\n\nEriksen M, Maximenko N, Thiel M, et al.: Plastic pollution in the South Pacific subtropical gyre. Mar. Pollut. Bull. 2013 Mar 15; 68(1–2): 71–76. PubMed Abstract \| Publisher Full Text\n\nSharma S, Sharma V, Chatterjee S: Microplastics in the Mediterranean Sea: Sources, Pollution Intensity, Sea Health, and Regulatory Policies. Front. Mar. Sci. 2021 May 5; 8: 494. Publisher Full Text\n\nAndrady AL: Persistence of plastic litter in the oceans. Mar. Anthropog. Litter. 2015 Jan 1 [cited 2022 Nov 26]; 29–56. Publisher Full Text\n\nvon der Kammer F , Ferguson PL, Holden PA, et al.: Analysis of engineered nanomaterials in complex matrices (environment and biota): General considerations and conceptual case studies. Environ. Toxicol. Chem. 2012 Jan 1 [cited 2022 Nov 26]; 31(1): 32–49. PubMed Abstract \| Publisher Full Text\n\nWagner M, Scherer C, Alvarez-Muñoz D, et al.: Microplastics in freshwater ecosystems: what we know and what we need to know. Environ. Sci. Eur. 2014 Dec 1 [cited 2022 Nov 26]; 26(1): 12–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinto J, Athanassiou A, Fragouli D: Effect of the porous structure of polymer foams on the remediation of oil spills. J. Phys. D. Appl. Phys. 2016 Mar 1 [cited 2022 Nov 26]; 49(14): 145601. Publisher Full Text\n\nKoelmans AA: Modeling the role of microplastics in bioaccumulation of organic chemicals to marine aquatic organisms. A critical review. Mar. Anthropog. Litter. 2015 Jan 1 [cited 2022 Nov 26]; 309–324. Publisher Full Text\n\nCai H, Xu EG, Du F, et al.: Analysis of environmental nanoplastics: Progress and challenges. Chem. Eng. J. 2021; 410: 128208. Publisher Full Text\n\nBessa F, Barría P, Neto JM, et al.: Occurrence of microplastics in commercial fish from a natural estuarine environment. Mar. Pollut. Bull. 2018 Mar 1 [cited 2022 Nov 26]; 128: 575–584. PubMed Abstract \| Publisher Full Text\n\nTamminga M, Hengstmann E, Fischer EK: Microplastic analysis in the South Funen Archipelago, Baltic Sea, implementing manta trawling and bulk sampling. Mar. Pollut. Bull. 2018 Mar 1 [cited 2022 Nov 26]; 128: 601–608. PubMed Abstract \| Publisher Full Text Reference Source\n\nZhou XX, Hao LT, Wang HYZ, et al.: Cloud-Point Extraction Combined with Thermal Degradation for Nanoplastic Analysis Using Pyrolysis Gas Chromatography-Mass Spectrometry. Anal. Chem. 2019 Feb 5 [cited 2022 Nov 26]; 91(3): 1785–1790. PubMed Abstract \| Publisher Full Text\n\nKoelmans AA, Besseling E, Shim WJ: Nanoplastics in the aquatic environment. Critical review. Mar. Anthropog. Litter. 2015 Jan 1 [cited 2022 Nov 26]; 325–340. Publisher Full Text\n\nFerreira I, Venâncio C, Lopes I, et al.: Nanoplastics and marine organisms: What has been studied? Environ. Toxicol. Pharmacol. 2019 Apr 1; 67: 1–7. Publisher Full Text\n\nGuterres SS, Alves MP, Pohlmann AR: Polymeric Nanoparticles, Nanospheres and Nanocapsules, for Cutaneous Applications. Drug Target Insights. 2007 Jan 9 [cited 2022 Nov 26]; 2: 117739280700200. Publisher Full Text\n\nFendall LS, Sewell MA. Contributing to marine pollution by washing your face: microplastics in facial cleansers. Mar. Pollut. Bull. 2009 Aug [cited 2022 Nov 26]; 58(8): 1225–8. PubMed Abstract \| Publisher Full Text\n\nLambert S, Wagner M: Characterisation of nanoplastics during the degradation of polystyrene. Chemosphere. 2016 Feb 1 [cited 2022 Nov 26]; 145: 265–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang H, Kuo YY, Gerecke AC, et al.: Co-release of hexabromocyclododecane (HBCD) and nano- and microparticles from thermal cutting of polystyrene foams. Environ. Sci. Technol. 2012 Oct 16 [cited 2022 Nov 26]; 46(20): 10990–10996. PubMed Abstract \| Publisher Full Text\n\nJang M, Shim WJ, Han GM, et al.: Widespread detection of a brominated flame retardant, hexabromocyclododecane, in expanded polystyrene marine debris and microplastics from South Korea and the Asia-Pacific coastal region. Environ. Pollut. 2017 Dec 1; 231: 785–794. PubMed Abstract \| Publisher Full Text\n\nSong YK, Hong SH, Jang M, et al.: Combined Effects of UV Exposure Duration and Mechanical Abrasion on Microplastic Fragmentation by Polymer Type. Environ. Sci. Technol. 2017 Apr 18 [cited 2022 Nov 26]; 51(8): 4368–4376. PubMed Abstract \| Publisher Full Text\n\nTalvitie J, Heinonen M, Pääkkönen JP, et al.: Do wastewater treatment plants act as a potential point source of microplastics? Preliminary study in the coastal Gulf of Finland, Baltic Sea. Water Sci. Technol. 2015 Nov 11; 72(9): 1495–1504. PubMed Abstract \| Publisher Full Text\n\nBrowne MA, Crump P, Niven SJ, et al.: Accumulation of microplastic on shorelines woldwide: Sources and sinks. Environ. Sci. Technol. 2011 Nov 1 [cited 2022 Nov 26]; 45(21): 9175–9179. PubMed Abstract \| Publisher Full Text\n\nvon der Kammer F , Ferguson PL, Holden PA, et al.: Analysis of engineered nanomaterials in complex matrices (environment and biota): general considerations and conceptual case studies. Environ. Toxicol. Chem. 2012 Jan [cited 2022 Dec 1]; 31(1): 32–49. PubMed Abstract \| Publisher Full Text\n\nLambert S, Wagner M: Formation of microscopic particles during the degradation of different polymers. Chemosphere. 2016; 161: 510–517. Publisher Full Text\n\nRhyner MN: The Coulter principle for analysis of subvisible particles in protein formulations. AAPS J. 2011 Mar [cited 2022 Dec 1]; 13(1): 54–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDemeule B, Messick S, Shire SJ, et al.: Characterization of particles in protein solutions: reaching the limits of current technologies. AAPS J. 2010 Dec [cited 2022 Dec 1]; 12(4): 708–715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNarhi L, Jiang Y, Cao S, et al.: A critical review of analytical methods for subvisible and visible particles. Curr. Pharm. Biotechnol. 2009 Jun 3 [cited 2022 Dec 1]; 10(4): 373–381. PubMed Abstract \| Publisher Full Text\n\nFilipe V, Hawe A, Jiskoot W: Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm. Res. 2010 May [cited 2022 Dec 1]; 27(5): 796–810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKramberger P, Ciringer M, Trancar A, et al.: Evaluation of nanoparticle tracking analysis for total virus particle determination. Virol. J. 2012 Nov 12 [cited 2022 Dec 1]; 9(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLambert S, Wagner M: Characterisation of nanoplastics during the degradation of polystyrene. Chemosphere. 2016 Feb 1 [cited 2022 Dec 1]; 145: 265–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg EL, Huerta Lwanga E, Eldridge SM, et al.: An overview of microplastic and nanoplastic pollution in agroecosystems. Sci. Total Environ. 2018 Jun 15; 627: 1377–1388. PubMed Abstract \| Publisher Full Text\n\nAlexy P, Anklam E, Emans T, et al.: Managing the analytical challenges related to micro- and nanoplastics in the environment and food: filling the knowledge gaps.2019 Jan 2 [cited 2022 Dec 1]; 37(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nPinto da Costa J, Reis V, Paço A, et al.: Micro (nano) plastics – Analytical challenges towards risk evaluation. TrAC Trends. Anal. Chem. 2019 Feb 1; 111: 173–184. Publisher Full Text\n\nHernandez LM, Yousefi N, Tufenkji N: Supporting Information Are There Nanoplastics in Your Personal Care Products?\n\nHernandez LM, Xu EG, Larsson HCE, et al.: Supplementary Information Plastic teabags release billions of microparticles and nanoparticles into tea.\n\nLu S, Zhu K, Song W, et al.: Impact of water chemistry on surface charge and aggregation of polystyrene microspheres suspensions. Sci. Total Environ. 2018 Jul 15; 630: 951–959. PubMed Abstract \| Publisher Full Text\n\nMagrì D, Sánchez-Moreno P, Caputo G, et al.: Laser Ablation as a Versatile Tool to Mimic Polyethylene Terephthalate Nanoplastic Pollutants: Characterization and Toxicology Assessment.\n\nMintenig SM, Bäuerlein PS, Koelmans AA, et al.: Closing the gap between small and smaller: towards a framework to analyse nano- and microplastics in aqueous environmental samples. Environ. Sci. Nano. 2018 Jul 13 [cited 2022 Dec 1]; 5(7): 1640–1649. Publisher Full Text Reference Source\n\nZhou X-X, Hao L-T, Wang H-Y-Z, et al.: Supporting Information Cloud Point Extraction Combined with Thermal Degradation for Nanoplastic Analysis Using Pyrolysis Gas Chromatography−Mass Spectrometry.\n\nDavranche M, Lory C, Le JC, et al.: Nanoplastics on the coast exposed to the North Atlantic Gyre: Evidence and traceability. NanoImpact. 2020 Oct 1; 20: 100262. Publisher Full Text\n\nWahl A, Le Juge C, Davranche M, et al.: Nanoplastic occurrence in a soil amended with plastic debris. Chemosphere. 2021 Jan 1; 262: 127784. PubMed Abstract \| Publisher Full Text\n\nLin Y, Huang X, Liu Q, et al.: Thermal fragmentation enhanced identification and quantification of polystyrene micro/nanoplastics in complex media. Talanta. 2020 Feb 1; 208: 120478. PubMed Abstract \| Publisher Full Text\n\nWu P, Tang Y, Cao G, et al.: Supporting Information for Determination of environmental micro-(nano-)plastics by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry Including Section I Environmental Section, Section II Method Validation, Section III with 3 Tab.\n\nTer Halle A, Jeanneau L, Martignac M, et al.: Nanoplastic in the North Atlantic subtropical gyre 1 Supplementary Materials 2 3 AUTHOR NAMES 4.\n\nKoelmans AA, Besseling E, Shim WJ: Nanoplastics in the aquatic environment. Critical review. Mar. Anthropog. Litter. 2015 Jan 1 [cited 2022 Dec 1]; 325–340. Publisher Full Text\n\nOliveira M, Almeida M: The why and how of micro (nano) plastic research. TrAC Trends Anal. Chem. 2019 May 1; 114: 196–201. Publisher Full Text\n\nGigault J, El Hadri H, Nguyen B, et al.: Nanoplastics are neither microplastics nor engineered nanoparticles. Nat. Nanotechnol. 2021; 16(5): 501–507. Publisher Full Text\n\nYin K, Wang Y, Zhao H, et al.: A comparative review of microplastics and nanoplastics: Toxicity hazards on digestive, reproductive and nervous system. Sci. Total Environ. 2021 Jun 20; 774: 145758. Publisher Full Text\n\nWu X, Pan J, Li M, et al.: Selective enrichment of bacterial pathogens by microplastic biofilm. Water Res. 2019 Nov 15 [cited 2021 Sep 12]; 165: 114979. PubMed Abstract \| Publisher Full Text\n\nRist S, Baun A, Hartmann NB: Ingestion of micro- and nanoplastics in Daphnia magna – Quantification of body burdens and assessment of feeding rates and reproduction. Environ. Pollut. 2017 Sep 1; 228: 398–407. PubMed Abstract \| Publisher Full Text\n\nJeong C-B, Won E-J, Kang H-M, et al.: Microplastic Size-Dependent Toxicity, Oxidative Stress Induction, and p-JNK and p-p38 Activation in the Monogonont Rotifer (Brachionus koreanus). Environ. Sci. Technol. 2016 Aug 16 [cited 2021 Sep 12]; 50(16): 8849–8857. PubMed Abstract \| Publisher Full Text\n\nTrevisan R, Voy C, Chen S, et al.: Nanoplastics Decrease the Toxicity of a Complex PAH Mixture but Impair Mitochondrial Energy Production in Developing Zebrafish. Environ. Sci. Technol. 2019 Jul 16 [cited 2021 Sep 12]; 53(14): 8405–8415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaylarde CC, Baptista Neto JA, da Fonseca EM : Nanoplastics in aquatic systems - are they more hazardous than microplastics? Environ. Pollut. 2021 Mar 1; 272: 115950. PubMed Abstract \| Publisher Full Text\n\nGe H, Yan Y, Wu D, et al.: Potential role of LINC00996 in colorectal cancer: a study based on data mining and bioinformatics. Onco Targets Ther. 2018 [cited 2022 Dec 1]; 11: 4845–4855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLai H, Liu X, Qu M: Nanoplastics and Human Health: Hazard Identification and Biointerface. Nanomaterials. 2022 Apr 1 [cited 2022 Dec 1]; 12(8). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernandez LM, Xu EG, Larsson HCE, et al.: Plastic Teabags Release Billions of Microparticles and Nanoparticles into Tea. Environ. Sci. Technol. 2019 Nov 5 [cited 2022 Dec 1]; 53(21): 12300–12310. PubMed Abstract \| Publisher Full Text\n\nYang D, Shi H, Li L, et al.: Microplastic Pollution in Table Salts from China. Environ. Sci. Technol. 2015 Oct 20 [cited 2021 Sep 12]; 49(22): 13622–13627. PubMed Abstract \| Publisher Full Text\n\nAlberts B, Johnson A, Lewis J, et al.: T Cells and MHC Proteins. Mol. Biol. Cell. 2008 [cited 2021 Sep 12]; 1569–1588. Reference Source\n\nCole M, Galloway TS: Ingestion of Nanoplastics and Microplastics by Pacific Oyster Larvae. Environ. Sci. Technol. 2015; 49(24): 14625–14632. PubMed Abstract \| Publisher Full Text\n\nJin Y, Xia J, Pan Z, et al.: Polystyrene microplastics induce microbiota dysbiosis and inflammation in the gut of adult zebrafish. Environ. Pollut. 2018 Apr 1 [cited 2022 Dec 2]; 235: 322–9. PubMed Abstract \| Publisher Full Text\n\nLi Y, Li M, Li Z, et al.: Effects of particle size and solution chemistry on Triclosan sorption on polystyrene microplastic. Chemosphere. 2019 Sep 1 [cited 2022 Dec 2]; 231: 308–14. PubMed Abstract \| Publisher Full Text\n\nLuo H, Xiang Y, He D, et al.: Leaching behavior of fluorescent additives from microplastics and the toxicity of leachate to Chlorella vulgaris. Sci. Total Environ. 2019 Aug 15 [cited 2022 Dec 2]; 678: 1–9. PubMed Abstract \| Publisher Full Text\n\nLu C, Kania PW, Buchmann K: Particle effects on fish gills: An immunogenetic approach for rainbow trout and zebrafish. Aquaculture. 2018 Feb 1; 484: 98–104. Publisher Full Text\n\nKarami A, Golieskardi A, Keong Choo C, et al.: The presence of microplastics in commercial salts from different countries. Sci. Report. 2017 Apr 6 [cited 2021 Sep 12]; 7(1): 1–11. Publisher Full Text Reference Source\n\nPrüst M, Meijer J, Westerink RHS: The plastic brain: neurotoxicity of micro- and nanoplastics. Part. Fibre Toxicol. 2020 Jun 8 [cited 2021 Sep 12]; 17(1): 16–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBergmann M, Gutow L, Klages M: Marine Anthropogenic Litter.\n\nWalczak AP, Kramer E, Hendriksen PJM, et al.: Translocation of differently sized and charged polystyrene nanoparticles in in vitro intestinal cell models of increasing complexity.2015 May 1 [cited 2022 Dec 2]; 9(4): 453–461. Publisher Full Text\n\nGe H, Yan Y, Wu D, et al.: Potential role of LINC00996 in colorectal cancer: a study based on data mining and bioinformatics. Onco Targets Ther. 2018 [cited 2022 Dec 2]; 11: 4845–4855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFood Safety Authority E: Presence of microplastics and nanoplastics in food, with particular focus on seafood. EFSA J. 2016 Jun 1 [cited 2022 Dec 2]; 14(6): e04501. Publisher Full Text\n\nLundqvist M, Stigler J, Elia G, et al.: Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. USA. 2008 Sep 23 [cited 2022 Dec 2]; 105(38): 14265–14270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTenzer S, Docter D, Kuharev J, et al.: Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat. Nanotechnol. 2013; 8(10): 772–781. PubMed Abstract \| Publisher Full Text\n\nLehner R, Weder C, Petri-Fink A, et al.: Emergence of Nanoplastic in the Environment and Possible Impact on Human Health. Environ. Sci. Technol. 2019 [cited 2022 Dec 2]; 53: 1748–1765. PubMed Abstract \| Publisher Full Text\n\nGopinath PM, Saranya V, Vijayakumar S, et al.: Assessment on interactive prospectives of nanoplastics with plasma proteins and the toxicological impacts of virgin, coronated and environmentally released-nanoplastics. Sci. Rep. 2019 Jun 20 [cited 2022 Dec 2]; 9(1): 1–15. Publisher Full Text Reference Source\n\nStapleton PA: Toxicological considerations of nano-sized plastics. AIMS Environ. Sci. 2019 [cited 2022 Dec 2]; 6(5): 367–378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrata JC, da Costa JP , Lopes I, et al.: Environmental exposure to microplastics: An overview on possible human health effects. Sci. Total Environ. 2020 Feb 1; 702: 134455. PubMed Abstract \| Publisher Full Text\n\nDris R, Gasperi J, Mirande C, et al.: A first overview of textile fibers, including microplastics, in indoor and outdoor environments. Environ. Pollut. 2017 Feb 1; 221: 453–458. PubMed Abstract \| Publisher Full Text\n\nDris R, Gasperi J, Saad M, et al.: Synthetic fibers in atmospheric fallout: A source of microplastics in the environment? Mar. Pollut. Bull. 2016 Mar 15; 104(1–2): 290–293. PubMed Abstract \| Publisher Full Text\n\nOhlwein S, Kappeler R, Kutlar Joss M, et al.: Health effects of ultrafine particles: a systematic literature review update of epidemiological evidence. Int. J. Public Health. 2019 Feb 21 [cited 2022 Dec 2]; 64(4): 547–559. PubMed Abstract \| Publisher Full Text\n\nGehr P, Bachofen M, Weibel ER: The normal human lung: ultrastructure and morphometric estimation of diffusion capacity. Respir. Physiol. 1978 [cited 2022 Dec 2]; 32(2): 121–40. PubMed Abstract \| Publisher Full Text\n\nYong CQY, Valiyaveettil S, Tang BL: Toxicity of Microplastics and Nanoplastics in Mammalian Systems. Int. J. Environ. Res. Public Health. 2020 Feb 26 [cited 2021 Sep 12]; 17(5): 1509. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nPrata JC, da Costa JP , Lopes I, et al.: Environmental exposure to microplastics: An overview on possible human health effects. Sci. Total Environ. 2020 Feb 1; 702: 134455. PubMed Abstract \| Publisher Full Text\n\nRahman A, Sarkar A, Yadav OP, et al.: Potential human health risks due to environmental exposure to nano- and microplastics and knowledge gaps: A scoping review. Sci. Total Environ. 2021 Feb 25; 757: 143872. PubMed Abstract \| Publisher Full Text\n\nXu M, Halimu G, Zhang Q, et al.: Internalization and toxicity: A preliminary study of effects of nanoplastic particles on human lung epithelial cell. Sci. Total Environ. 2019 Dec 1; 694: 133794. PubMed Abstract \| Publisher Full Text\n\nShi Q, Tang J, Wang L, et al.: Combined cytotoxicity of polystyrene nanoplastics and phthalate esters on human lung epithelial A549 cells and its mechanism. Ecotoxicol. Environ. Saf. 2021 Apr 15; 213: 112041. PubMed Abstract \| Publisher Full Text\n\nRist S, Carney Almroth B, Hartmann NB, et al.: A critical perspective on early communications concerning human health aspects of microplastics. Sci. Total Environ. 2018 Jun 1; 626: 720–726. PubMed Abstract \| Publisher Full Text\n\nPrata JC: Airborne microplastics: Consequences to human health? Environ. Pollut. 2018 Mar 1; 234: 115–126. PubMed Abstract \| Publisher Full Text\n\nBouwstra J, Pilgram G, Gooris G, et al.: New Aspects of the Skin Barrier Organization. Skin Pharmacol. Physiol. 2001 [cited 2021 Sep 12]; 14(Suppl. 1): 52–62. Publisher Full Text Reference Source\n\nSchneider M, Stracke F, Hansen S, et al.: Nanoparticles and their interactions with the dermal barrier. Dermatoendocrinol. 2009 Jul [cited 2021 Sep 12]; 1(4): 197–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvarez-Román R, Naik A, Kalia YN, et al.: Skin penetration and distribution of polymeric nanoparticles. J. Control. Release. 2004 Sep 14; 99(1): 53–62. Publisher Full Text\n\nCampbell CSJ, Contreras-Rojas LR, Delgado-Charro MB, et al.: Objective assessment of nanoparticle disposition in mammalian skin after topical exposure. J. Control. Release. 2012 Aug 20; 162(1): 201–207. PubMed Abstract \| Publisher Full Text\n\nVogt A, Combadiere B, Hadam S, et al.: 40 nm, but not 750 or 1,500 nm, Nanoparticles Enter Epidermal CD1a+ Cells after Transcutaneous Application on Human Skin. J. Invest. Dermatol. 2006 Jun 1; 126(6): 1316–1322. Publisher Full Text\n\nBiniek K, Levi K, Dauskardt RH: Solar UV radiation reduces the barrier function of human skin. Proc. Natl. Acad. Sci. USA. 2012 Oct 16 [cited 2022 Dec 2]; 109(42): 17111–17116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossi G, Barnoud J, Monticelli L: Polystyrene nanoparticles perturb lipid membranes. J. Phys. Chem. Lett. 2014 Jan 2 [cited 2022 Dec 2]; 5(1): 241–246. PubMed Abstract \| Publisher Full Text\n\nSalvati A, Pitek AS, Monopoli MP, et al.: Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 2013 Jan 20 [cited 2022 Dec 2]; 8(2): 137–143. PubMed Abstract \| Publisher Full Text Reference Source\n\nAderem A, Underhill DM: Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 1999; 17: 593–623. Publisher Full Text\n\nSwanson JA, Yoshida S: Macropinocytosis. Encycl. Cell Biol. 2016; 2: 758–765.\n\nTakei K, Haucke V: Clathrin-mediated endocytosis: Membrane factors pull the trigger. Trends Cell Biol. 2001 Sep 1; 11(9): 385–391. PubMed Abstract \| Publisher Full Text\n\nKovtun O, Tillu VA, Ariotti N, et al.: Cavin family proteins and the assembly of caveolae. J. Cell Sci. 2015; 128(7): 1269–1278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFiorentino I, Gualtieri R, Barbato V, et al.: Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures. Exp. Cell Res. 2015 Jan 15; 330(2): 240–247. PubMed Abstract \| Publisher Full Text\n\ndos Santos T , Varela J, Lynch I, et al.: Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines. PLoS One. 2011 Sep 19 [cited 2022 Dec 2]; 6(9): e24438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYee MSL, Hii LW, Looi CK, et al.: Impact of Microplastics and Nanoplastics on Human Health. Nano. 2021 Feb 1 [cited 2022 Dec 2]; 11(2): 1–23. Publisher Full Text \| Free Full Text\n\nDing J, Zhang S, Razanajatovo RM, et al.: Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environ. Pollut. 2018 Jul 1; 238: 1–9. PubMed Abstract \| Publisher Full Text\n\nGeiser M, Rothen-Rutishauser B, Kapp N, et al.: Ultrafine Particles Cross Cellular Membranes by Nonphagocytic Mechanisms in Lungs and in Cultured Cells. Environ. Health Perspect. 2005 Nov [cited 2022 Dec 5]; 113(11): 1555–1560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMattsson K, Johnson EV, Malmendal A, et al.: Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain. Sci. Report. 2017 Sep 13 [cited 2022 Dec 5]; 7(1): 1–7. Publisher Full Text Reference Source\n\nKrug HF, Wick P: Nanotoxicology: An interdisciplinary challenge. Angew. Chem. Int. Ed. 2011 Feb 7; 50(6): 1260–1278. PubMed Abstract \| Publisher Full Text\n\nDing J, Huang Y, Liu S, et al.: Toxicological effects of nano- and micro-polystyrene plastics on red tilapia: Are larger plastic particles more harmless? J. Hazard. Mater. 2020 Sep 5; 396: 122693. PubMed Abstract \| Publisher Full Text\n\nWan Z, Wang C, Zhou J, et al.: Effects of polystyrene microplastics on the composition of the microbiome and metabolism in larval zebrafish. Chemosphere. 2019 Feb 1; 217: 646–658. PubMed Abstract \| Publisher Full Text\n\nChen Q, Yin D, Jia Y, et al.: Enhanced uptake of BPA in the presence of nanoplastics can lead to neurotoxic effects in adult zebrafish. Sci. Total Environ. 2017 Dec 31; 609: 1312–1321. PubMed Abstract \| Publisher Full Text\n\nChen Q, Gundlach M, Yang S, et al.: Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 2017 Apr 15; 584-585: 1022–1031. Publisher Full Text\n\nSultana R, Perluigi M, Butterfield DA: Lipid Peroxidation Triggers Neurodegeneration: A Redox Proteomics View into the Alzheimer Disease Brain. Free Radic. Biol. Med. 2013 [cited 2022 Dec 5]; 62: 157–169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShichiri M: The role of lipid peroxidation in neurological disorders. J. Clin. Biochem. Nutr. 2014 [cited 2022 Dec 5]; 54(3): 151–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaso OV, Philippou A, Moustogiannis A, et al.: Lipid peroxidation products and their role in neurodegenerative diseases. Ann Res Hosp. 2019 Jan 16 [cited 2022 Dec 5]; 3: 2–2. Publisher Full Text Reference Source\n\nBarboza LGA, Lopes C, Oliveira P, et al.: Microplastics in wild fish from North East Atlantic Ocean and its potential for causing neurotoxic effects, lipid oxidative damage, and human health risks associated with ingestion exposure. Sci. Total Environ. 2020 May 15; 717: 134625. PubMed Abstract \| Publisher Full Text\n\nTrang A, Khandhar PB: Physiology, Acetylcholinesterase. StatPearls; 2022 May 8 [cited 2022 Dec 5]. Reference Source\n\nMcGrath P, Li CQ: Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov. Today. 2008 May 1; 13(9–10): 394–401. PubMed Abstract \| Publisher Full Text\n\nMattsson K, Ekvall MT, Hansson LA, et al.: Altered behavior, physiology, and metabolism in fish exposed to polystyrene nanoparticles. Environ. Sci. Technol. 2015 Jan 6 [cited 2022 Dec 5]; 49(1): 553–561. PubMed Abstract \| Publisher Full Text\n\nHoelting L, Scheinhardt B, Bondarenko O, et al.: A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles. Arch. Toxicol. 2012 Dec 2 [cited 2021 Sep 12]; 87(4): 721–733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLasky JL, Wu H: Notch Signaling, Brain Development, and Human Disease. Pediatr. Res. 2005 [cited 2021 Sep 12]; 57(7): 104R–109R. Publisher Full Text Reference Source\n\nCastro DS, Martynoga B, Parras C, et al.: A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. Genes Dev. 2011 May 1 [cited 2021 Sep 12]; 25(9): 930–945. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nBan M, Shimoda R, Chen J: Investigation of nanoplastic cytotoxicity using SH-SY5Y human neuroblastoma cells and polystyrene nanoparticles. Toxicol Vitr. 2021 Oct 1; 76: 105225. PubMed Abstract \| Publisher Full Text\n\nJung BK, Han SW, Park SH, et al.: Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain. Neurotoxicology. 2020 Dec 1; 81: 189–196. PubMed Abstract \| Publisher Full Text\n\nRafiee M, Dargahi L, Eslami A, et al.: Neurobehavioral assessment of rats exposed to pristine polystyrene nanoplastics upon oral exposure. Chemosphere. 2018 Feb 1; 193: 745–753. PubMed Abstract \| Publisher Full Text\n\nLiu Q, Chen C, Li M, et al.: Neurodevelopmental Toxicity of Polystyrene Nanoplastics in Caenorhabditis elegans and the Regulating Effect of Presenilin. ACS. Omega. 2020 Dec 29 [cited 2021 Sep 12]; 5(51): 33170–33177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaró I, Perini A, Torreblanca A, et al.: Time-dependent effects of polystyrene nanoparticles in brine shrimp Artemia franciscana at physiological, biochemical and molecular levels. Sci. Total Environ. 2019; 675: 570–580. PubMed Abstract \| Publisher Full Text\n\nWindheim J, Colombo L, Battajni NC, et al.: Micro- and Nanoplastics’ Effects on Protein Folding and Amyloidosis. Int. J. Mol. Sci. 2022 Sep 7 [cited 2022 Dec 5]; 23(18): 10329. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nGopinath PM, Saranya V, Vijayakumar S, et al.: Assessment on interactive prospectives of nanoplastics with plasma proteins and the toxicological impacts of virgin, coronated and environmentally released-nanoplastics. Sci. Rep. 2019 Jun 20 [cited 2022 Dec 5]; 9(1): 1–15. Reference Source\n\nHollóczki O, Gehrke S: Nanoplastics can change the secondary structure of proteins. Sci. Rep. 2019 Nov 5 [cited 2022 Dec 5]; 9(1): 1–7. Publisher Full Text Reference Source\n\nLinse S, Cabaleiro-Lago C, Xue WF, et al.: Nucleation of protein fibrillation by nanoparticles. Proc. Natl. Acad. Sci. USA. 2007 May 22 [cited 2022 Dec 5]; 104(21): 8691–8696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSavva GM, Wharton SB, Ince PG, et al.: Age, Neuropathology, and Dementia. N. Engl. J. Med. 2009 May 28; 360(22): 2302–2309. Publisher Full Text\n\nLiang B, Huang Y, Zhong Y, et al.: Brain single-nucleus transcriptomics highlights that polystyrene nanoplastics potentially induce Parkinson’s disease-like neurodegeneration by causing energy metabolism disorders in mice. J. Hazard. Mater. 2022 May 15; 430: 128459. PubMed Abstract \| Publisher Full Text" }	[ { "id": "315923", "date": "30 Aug 2024", "name": "Yankai Xia", "expertise": [ "Reviewer Expertise environment and health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review addresses the increasingly relevant topic of the neurotoxic potential of nanoplastics (NPs) in the context of escalating plastic pollution, effectively summarizing key findings from the literature with an emphasis on the various exposure routes and associated risks. However, the manuscript would benefit from a more comprehensive synthesis of the existing literature, particularly in addressing the inconsistencies and gaps in current research, while also providing a clearer articulation of the limitations of current research methodologies and offering suggestions for future studies. Additionally, a discussion of the broader implications for public health and potential regulatory frameworks would strengthen the manuscript's contribution to the field. Overall, the review could be further improved by deepening the analysis of existing studies and providing a more critical perspective on the current state of knowledge. I recommend that the authors consider resubmitting after making significant improvements.\nMajor comments\nWhile the review discusses various detection and quantification methods for NPs, a more detailed critique of the limitations of these methodologies is needed. This should include an examination of the challenges related to detecting NPs in environmental samples versus laboratory conditions, as well as the implications these limitations have for interpreting research findings. The manuscript needs a more critical analysis of key research gaps, especially concerning the inconsistencies in findings related to the mechanisms of NP-induced neurotoxicity. Strengthening this section with a more detailed comparison of the outcomes across different experimental models and conditions would greatly enhance the review's contribution. The discussion on the mechanisms of NP-induced neurotoxicity is crucial. For instance, exploring the specific biochemical pathways through which NPs interact with cellular components at a molecular level would provide a more comprehensive understanding. The role of protein corona formation in neurotoxicity, mentioned towards the end, should be integrated earlier in the manuscript to establish a clear connection between NP exposure and neurodegenerative diseases. While the manuscript covers many trending topics, it often treats them in isolation, which leads to a lack of coherence. An integrated approach that links these topics and demonstrates their interconnections would greatly improve the flow and continuity of the review.\nMinor comments\nThe manuscript relies heavily on older studies, with relatively few references from the past three years. Incorporating more recent studies will ensure that the review reflects the current state of research and provides a comprehensive overview of the field. In some sections, particularly those discussing in vivo studies, the outcomes are not always clearly connected to the broader implications for neurotoxicity. It would be helpful to more explicitly link the results of these studies to the potential mechanisms of NP-induced neurotoxicity and their relevance to human health. The conclusion primarily restates the findings discussed throughout the review but does not provide a comprehensive synthesis of the key takeaways. The summary of neurotoxicity of NPs in different models presented in Table 1 is not comprehensive and should be thoroughly enumerated. The language of the manuscript should be polished.\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? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] }, { "id": "332717", "date": "25 Nov 2024", "name": "Amitava Mukherjee", "expertise": [ "Reviewer Expertise Environemntal Science", "Micro nano Plastics", "Toxicology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review presents an exhaustive coverage of the neurotoxic effects of nanoplastics. The authors have done a commendable job of collecting literature and making a balanced presentation. However, I suggest the following points.\n1. Introduction: The introduction is rather about the issues with plastic pollution, kindly introduce the importance and relevance of the neurotoxicity of the plastics here and also add a brief outline of the topics covered in the Review. Given that already a sizeable number of reviews are available on the topic of plastic pollution, please make this part brief and bring out the title of the work, \"neurotoxicity\" here.\n2. Under Nanopalstics please revise the discussion on sources of the NPs relevant to human uptake and toxicity. Please connect this part with the main thread of the review. This is also much discussed in the literature already, and so with appropriate citations, the authors can shorten the description here.\nIn the detection and quantification clearly distinguish and discuss the in vitro and in vivo detection and challenges associated briefly.\nThe differences between MPs and Nps is a misfit in the review and out of context, in the introduction section itself one or two lines can be added with specific references for interested readers.\n3. In the \"potential routes of NP exposure to Humans\" please avoid adding mechanisms of interaction/effects in this section, stick to the sources.\nIntracellular fate and bio-corona again may not fit well as a separate section, please integrate them briefly into the section on \"uptake\" and make their relevance clear for neurotoxicity effects.\n4. Instead of sensitivity of the brain to oxidative stress discuss the various modes of action of the plastic particles mentioning why ROS is considered predominant one.. add relevance to plastic particles here briefly explain the effects of multiple chemical types, and possibly leaching of additives briefly.\n5. Looking at the length of the review roughly 30% is covered on neurotoxicity, please elaborate on mechanisms of action, effects of plastic types, and size-based effects of nano plastics with specifics on neurotoxicity. I assume the literature is replete with studies with polystyrene NPs but please see whether the effects of other plastic types can be added and the effects of weathered or environment-derived ones.\n6. Add a section on current gaps and challenges in these studies.\n7. Please add a section on methods of review, year range selected, inclusion/exclusion criteria adopted search engines used, and so on. Please add this after the introduction section. This is an important miss in the article.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-793
https://f1000research.com/articles/7-1902/v1	06 Dec 18	{ "type": "Software Tool Article", "title": "ESforRPD2: Expert System for Rice Plant Disease Diagnosis", "authors": [ "Fahrul Agus", "Muh. Ihsan", "Dyna Marisa Khairina", "Krishna Purnawan Candra", "Muh. Ihsan", "Dyna Marisa Khairina", "Krishna Purnawan Candra" ], "abstract": "One of the factors causing rice production disturbance in Indonesia is the lack of knowledge of farmers on early symptoms of rice plant diseases. These diseases are increasingly rampant because of the lack of experts. This study aimed to overcome this problem by providing an Expert System that helps farmers to make early diagnosis of rice plant diseases. Data of rice plant pests and diseases in 2016 were taken from Samarinda, East Kalimantan, Indonesia using an in-depth survey, and rice experts from the Department of Food Crops and Horticulture of East Kalimantan Province were recruited for the project. The Expert System for Rice Plant Disease Diagnosis, ESforRPD2, was developed based on the pest and disease experiences of the rice experts, and uses a Waterfall Paradigm and Unified Modelling Language. This Expert System can detect 48 symptoms and 8 types of diseases of rice plants from 16 data tests with an accuracy of 87.5%. ESforRPD2 is available in Indonesian at: http://esforrpd2.blog.unmul.ac.id", "keywords": [ "Expert System", "Rice Plant Disease", "Waterfall", "Unified Modelling Language" ], "content": "Introduction\n\nCorrect diagnosis of symptoms in rice plant diseases, caused by bacteria, nematodes, fungi, phythoplasmal and viruses1–4, is very critical in supporting the productivity of rice plants. However, many regions in Indonesia have a huge problem because of a limited number of rice plant pathologists. The large plantation area of rice plants is also a problem due to logistical issues when visiting these sites, leading to difficulty obtaining disease evidence.\n\nAlong with other rapid technological developments, a technology known as Expert System (ES)5–8 has been developed to solve health9–12, education13, and business14, including agriculture15,16, problems. ES is usually designed for a specific condition, i.e. variables of climate in cases of agriculture. This article proposes a new software based on ES for the diagnosis of disease in rice plants in the Samarinda region, Indonesia. Waterfall Paradigm applied in designing this ES. The prototype, Expert System for Rice Plant Disease Diagnosis (ESforRPD2) is available at: http://esforrpd2.blog.unmul.ac.id.\n\n\nMethods\n\nThe ES of rice plant disease diagnosis was designed to help farmers and agricultural officials to diagnose rice plant diseases occurring in the Samarinda region, East Kalimantan province, Indonesia. Rice plant experts were recruited from the Seed Technology Development Division at the Department of Food Crops and Horticulture of East Kalimantan Province and from the Department of Agro-eco-technology of Agricultural Faculty of Mulawarman University (one expert from each). The experts were the primary source for information on rice plant symptoms and diseases. The two rice plant experts have experience in diagnosing rice plant disease in the region of East Kalimantan Province for 20 years. Symptoms, diseases and their relationships (and their ranked importance) were derived from the experts by questionnaire (Supplementary File 1). This information was then used to construct the knowledge base for building the ES software.\n\nThe ES software was developed using the Waterfall paradigm as recommended by Sommerville17 using five stages, i.e. (i) planning and requirement, (ii) analysis and software design, (iii) implementation and unit testing, (iv) integration and (v) system testing and operation and maintenance. ES architecture consists of three parts, namely the user interface, the inference engine and the knowledge base as proposed by Lucas and van der Gaag7. The user interface is used as a consulting interface in order to obtain knowledge and advice from the ES, which would be like consulting an expert. In this ES, the inference engine works as a consultation system in processing input data to build a diagnosis based on the knowledge base developed.\n\nThe implementation of the ESforRPD2 application is based on Unified Modelling Language (Figure 1) as proposed by Sommerville17, which consists of use case diagrams, activity diagrams, and class diagrams.\n\na. Use case diagram of user. b. Use case diagram of expert.\n\nWe constructed two types of “Use case diagram”, namely “Use case for user” consisting of four cases (Article, Consulting, Choose Symptoms and Consulting Result); and “Use case for expert” consisting of three cases (Symptoms, Diseases, and Relation). The use case describes the functions of the ES interacting with user and expert. The activity diagram illustrates the flow of various activities being designed in the ES, i.e. how the flow starts, the decision that might occur, and the flow end. The activity diagram also describes parallel processes that might occur in some executions. In this ES, we build four data stores (Expert, Symptoms, Relation, and Diagnosis) in the class diagram. The ESforRPD2 application uses four datasets, namely disease- and symptoms-data, knowledge base, and symptoms-disease-weight relationships table (Dataset 2). The construction of decision trees and forward-chaining tracing for diagnosing of rice plant diseases in the ES is shown in Figure 2.\n\nESforRPD2 is the first version of ES (only in Indonesian) to make it user-friendly for Indonesian users. Users use a consultation page to choose the symptoms of the rice plant. The ES performs the calculation process to obtain the trust level using the Dempster-Shafer method18. The user page (Figure 3a) is the main web page for users without logging in. In the user page, there is also a home menu that displays articles about ES, rice plant diseases, and the Dempster-Shafer method. The consultation page starts the user consultation about the disease of rice plants (Figure 3b). The ES will provide an output as a display showing the symptoms, diagnosis of disease and the confidence level (Figure 3c).\n\nThe ESforRPD2 application is developed using CPU with specifications of Intel Core i3, 4GB RAM, and 300GB HDD. The same specification of CPU is needed to operate this application.\n\n\nUses case\n\nThe ESforRPD2 application was tested applying symptom-data inputs by clicking the symptoms selected (Figure 5b). In a single test using the case of four symptom-data inputs selected, namely (i) Spots on leaf midrib, (ii) Little spots are dark brown or slightly purple rounded shape, (iii) Spots on oval-shaped leaves and evenly distributed on the leaf surface, (iv) The size of spots is 2–10 mm long and 1 mm wide, a display of diagnosis page (Figure 3c) will appear following clicking of the “submit diagnose” button. The diagnosis page shows the confidence level. In this case test, the ES gave the accuracy of disease type detection of 91%.\n\n16 tests in row were conducted using randomly selected symptoms by user in the ES. The results were approved by the two experts. In total, 14 diagnosis (87.5%) of the 16 results showed by the ES were justified by the two experts (Table 1).\n\n\nDiscussion\n\nThe ESforRPD2 application is showing good reliability. By applying 16 tests, the ESforRPD2 showed a level of performance of 87.5% (Table 1) following justification to two rice plants diseases experts. The performance of the ESforRPD2 during validation was the expected high-performance level of plant diseases diagnosis by the expert system. This performance is much higher than the performance of ES for Chili pepper pest diagnosis invented by Agus et al.16. However other Expert System could show excellent performance of 98.38%19, this evidence advice that the performance of ESforRPD2 could be improved in the next study.\n\nCurrently, ESforRPD2 has only been tested with data from the Samarinda region. In a future study, we will use data from other regions of East Kalimantan, which have the same climate (tropical rainforest) and soil character as the Samarinda region. In addition, we will test data from other regions in Indonesia, which have a different climate. Newbery et al.20 showed that different climate conditions affect symptoms of arable crop disease; therefore, the ESforRPD2 will need continuous evaluation because climate change effects21.\n\n\nConsent\n\nWritten informed consent was obtained from the two experts for participation in the study.\n\n\nSoftware availability\n\nSoftware application is available from: http://esforrpd2.blog.unmul.ac.id.\n\nSource code: https://github.com/fahrulagus/paper.\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.149064122\n\nLicense: GNU GPL v3.0\n\n\nData availability\n\nZenodo: Knowledge base for rice plant disease diagnosis, https://doi.org/10.5281/zenodo.149065823\n\nZenodo: Dataset for rice plant diseases expert interview, http://doi.org/10.5281/zenodo.195338324", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors are grateful to both experts in this research, the Rector of Mulawarman University and Islamic Development Bank Project.\n\n\nReferences\n\nJagan Mohan K, Balasubramanian M, Palanivel S: Detection and Recognition of Diseases from Paddy Plant Leaf Images. Int J Comput Appl. 2016; 144(12): 34–41. Reference Source\n\nChapuis E, Besnard G, Andrianasetra S, et al.: First report of the root-knot nematode (Meloidogyne graminicola) in Madagascar rice fields. Australas Plant Dis Notes. 2016; 11(1): 32. Publisher Full Text\n\nQudsia H, Akhter M, Riaz A, et al.: Comparative Efficacy of Different Chemical Treatments for Paddy Blast, Brown Leaf Spot and Bacterial Leaf Blight Diseases in Rice (Oryza Sativa L.). Appl Microbiol Open Access. 2017; 3(3). Publisher Full Text\n\nKulmitra AK, Sahu N, Kumar VBS, et al.: In vitro evaluation of bio-agents against Pyricularia oryzae (Cav.) causing rice blast disease. Agric Sci Dig - A Res J. 2017; 37(3): 98–101. Publisher Full Text\n\nTodd BS: An Introduction to Expert Systems. Issue 95, Oxford University Computing Laboratory, Programming Research Group; 1992. Reference Source\n\nTurban E, Frenzel LE: Expert Systems and Applied Artificial Intelligence. Prentice Hall Professional Technical Reference; 1992. Reference Source\n\nLucas PJF, van der Gaag LC: Principles of Expert Systems. Amsterdam: Addison-Wesley; 1991. Reference Source\n\nGaines BR: Designing Expert Systems for Usability. Knowl Creat Diffus Util. 1986; 1–40. Reference Source\n\nGudu J, Gichoya D, Nyongesa P, et al.: Development of a medical expert system as an expert knowledge sharing tool on diagnosis and treatment of hypertension in pregnancy. Int J Biosci Biochem Bioinforma. 2012; 2(5): 297–300. Publisher Full Text\n\nAbu Naser SS, Ola AZA: An expert system for diagnosing eye diseases using clips. Theor Appl Inf Technol. 2008; 923–930. Reference Source\n\nAyangbekun OJ, Jimoh IA: Expert System for Diagnosis Neurodegenerative Diseases. Int J Comput Inf Technol. 2015; 4(4): 694–698. Reference Source\n\nAyangbekun OJ, Bankole FO: An Expert System for Diagnosis of Blood Disorder. Int J Comput Appl. 2014; 100(7): 975–8887.\n\nDivayana DGH: Utilization of cse-ucla model in evaluating of digital library program based on expert system at universitas teknologi indonesia: A model for evaluating of information technology-based education services. J Theor Appl Inf Technol. 2017; 95(15): 3585–3596. Reference Source\n\nArias-Aranda D, Castro JL, Navarro M, et al.: A fuzzy expert system for business management. Expert Syst Appl. 2010; 37(12): 7570–7580. Publisher Full Text\n\nIhsan M, Agus F, Khairina DM: Penerapan Metode Dempster Shafer Untuk Sistem Deteksi Penyakit Tanaman Padi. Prosiding Seminar Ilmu Komputer dan Teknologi Informasi. 2017; 2(1). Reference Source\n\nAgus F, Wulandari HE, Astuti IF: Expert System With Certainty Factor For Early Diagnosis Of Red Chili Peppers Diseases. JAIS. 2017; 2(2): 52–66. Reference Source\n\nSommerville I: Software Engineering. 10th Edition. Pearson; 2016. Reference Source\n\nMaseleno A, Mahmud Hasan M: Skin infection detection using Dempster-Shafer theory. In: 2012 International Conference on Informatics, Electronics and Vision, ICIEV 2012. 2012. Publisher Full Text\n\nSabzi S, Abbaspour-Gilandeh Y, García-Mateos G: A fast and accurate expert system for weed identification in potato crops using metaheuristic algorithms. Comput Ind. 2018; 98: 80–89. Publisher Full Text\n\nNewbery F, Qi A, Fitt BD: Modelling impacts of climate change on arable crop diseases: progress, challenges and applications. Curr Opin Plant Biol. 2016; 32: 101–109. PubMed Abstract \| Publisher Full Text\n\nXu C, Wu W, Ge Q: Impact assessment of climate change on rice yields using the ORYZA model in the Sichuan Basin, China. Int J Clim. 2018; 38(7): 2922–2939. Publisher Full Text\n\nfahrulagus,, Fajar MI: fahrulagus/paper: ESforRPD2 (Version V.10). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1490641\n\nAgus F, Ihsan M, Khairina DM, et al.: Knowledge base for rice plant disease diagnosis [Data set]. Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1490658\n\nAgus F, Ihsan M, Khairina DM, et al.: Dataset for rice plant diseases expert interview [Data set]. F1000Research. Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1953383" }	[ { "id": "43488", "date": "24 Jan 2019", "name": "Yi Fang", "expertise": [ "Reviewer Expertise electrochemistry", "plant diseases", "biosensors", "sensor" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 applied Expert System (ES) for rice plant disease and diagnosis; the background information and introduction is sufficient and well organized. The entire manuscript is also presented well. However, the author used the word “accuracy” which is not quite a scientific term. If “accuracy” is defined as sensitivity of the method, how about the specificity of the method? If a method has low specificity, it may not be able to solve the problem from false positive. For other comments, please see below:\nPage 1: Change “is the lack of knowledge of farmers on early symptoms…” to “is that farmers lack of knowledge of early symptoms…”.\n\nPage 1: “accuracy of 87.5%” accuracy is not a scientific terminology, do you refer to sensitivity or specificity?\n\nPage 3: change “… education, and business, including agriculture, problems” to “…education, business, and agriculture problems.”\n\nPage 3: please change to “Waterfall Paradigm was applied in designing this ES.”\n\nPage 5: “In this case test, the ES gave the accuracy of disease type detection of 91%”. Do you refer to sensitivity? Please also try to apply this comment to the other “accuracy” you mentioned in the manuscript.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4418", "date": "12 Feb 2019", "name": "Fahrul Agus", "role": "Author Response", "response": "We agree with your judgment regarding the term of accuracy. We meant the accuracy is the sensitivity, for that reason we change the term accuracy to sensitivity. Regarding the term of specificity, we explain that this system has high specificity for rice plants because all data used in constructing the algorithm were collected specifically for rice plant diseases." } ] } ]	1	https://f1000research.com/articles/7-1902
https://f1000research.com/articles/13-790/v1	11 Jul 24	{ "type": "Systematic Review", "title": "Prevalence of musculoskeletal symptoms from online learning during the COVID-19 epidemic: a systematic review and meta-analysis", "authors": [ "Tanawat Gotum", "Orawan Keeratisiroj", "Wutthichai Jariya", "Tanawat Gotum", "Wutthichai Jariya" ], "abstract": "Purpose The objective of this research was to assess the prevalence of musculoskeletal symptoms in online students.\n\nMaterials and methods A systematic review and meta-analysis were performed by searching the PubMed, Cochrane Library, SCOPUS, Web of Science, ScienceDirect, ProQuest, CINAHL plus with full text, and Wiley InterScience databases. A total of 3,749 studies were identified between January 2020 and December 2023. The Joanna Briggs Tool for studies reporting prevalence was used to assess the quality of studies. Jamovi 2.4 was used in the meta-analysis.\n\nResults Sixteen studies were included and used for the meta-analysis. There were 6 studies of high quality, 9 studies of medium quality and 1 study of low quality. The areas with the highest prevalence of musculoskeletal pain were the neck (51%, 95% CI = 36–66%), lower back (51%, 95% CI = 42–59%) and shoulder (36%, 95% CI = 26–47%).\n\nConclusions The shift to online learning during the COVID-19 pandemic has emerged as a potential factor influencing musculoskeletal pain in students. Educational institutions should study the duration of online learning that begins to impact student injury outcomes.", "keywords": [ "COVID-19", "Meta-analysis", "Musculoskeletal pain", "Online learning", "Student", "Systematic review" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) pandemic has emerged as a global health crisis due to its widespread transmission and ease of airborne contagion. With more than 703 million confirmed cases and 774 million fatalities worldwide as of December 31, 2023,1 countries around the globe have implemented stringent measures to curb the spread of the virus. Among these measures, city lockdowns have emerged as a prominent strategy to contain the COVID-19 pandemic. However, these lockdowns have significantly impacted the daily lives of individuals worldwide.2 City lockdowns or lockdowns restrict public movement, border crossings, and dine-in services at restaurants, fundamentally altering people’s daily routines globally. The impact extends to both work and education, with working professionals transitioning to remote work arrangements and students shifting to online learning platforms.3\n\nAs the COVID-19 pandemic continues to spread unabated across many countries, including Thailand, educational institutions have adopted online learning strategies to mitigate the risk of infection.4 Online learning has significantly impacted students’ daily lives, as they engage in virtual instruction via electronic devices connected to the internet. Instead of traditional classroom settings, students spend time in front of computers, smartphones, or tablets, typically for 6-8 hours per day, 3-5 days per week. This shift to remote learning has led to a substantial decrease in physical activity among students. A study by the Thai Center for Knowledge Development on Physical Activity revealed that even in normal circumstances, Thai children engage in sedentary behavior for more than 13 hours per day. However, the COVID-19 pandemic has exacerbated this issue, with sedentary behavior increasing to 14 hours per day for Thai children. This sedentary lifestyle is largely attributed to the shift toward online learning and prolonged screen time.5 The World Health Organization recommends that children engage in at least 60 minutes of physical activity daily.6 Data collected in Thailand over the past decade indicate that only 26% of Thai children meet these physical activity guidelines. Notably, during the COVID-19 pandemic, since 2020, the proportion of Thai children meeting the recommended physical activity standards has further declined to 17%.5 International studies have also documented a decline in physical activity among university students and younger people during the COVID-19 pandemic. A study in Indonesia revealed that university students’ physical activity levels decreased to an average of 60.92 minutes per week during the pandemic.7 Similarly, a German study involving 1,711 children and adolescents revealed a reduction in sports participation among those aged 4-17 years during the pandemic.8 Furthermore, an Italian study revealed a decrease in physical activity among university students during the pandemic, with lower levels of physical activity associated with an increased risk of musculoskeletal pain onset and worsening pain symptoms.9\n\nProlonged physical inactivity or maintaining a static posture for extended periods can lead to musculoskeletal problems, including fatigue, localized pain, and other symptoms.10 This issue extends beyond students and affects teachers as well. Research investigating musculoskeletal disorders (MSDs) among students engaged in online learning during the COVID-19 pandemic has revealed an increased prevalence of muscle aches and pains among this population. A study involving a sample of 261 students who transitioned to online learning during the pandemic revealed that 80% of participants reported experiencing headaches, eye strain, and neck pain following online classes.11 A study in Iran compared musculoskeletal symptoms among 220 faculty members at Yazd University before and during the COVID-19 pandemic using the Standardized Nordic Questionnaire. The findings revealed a statistically significant increase in musculoskeletal complaints among university faculty during the pandemic.12 MSDs are a prevalent global health concern. A study in the United States involving 654 university students reported a 12.5% prevalence of joint disorders among participants.13 Another study among computer-using university students reported that 23% of respondents had used medication to manage musculoskeletal symptoms.14 A South African study involving 145 participants revealed a high prevalence of musculoskeletal pain (89.7%) among university students.15 Additionally, a Chinese study among first-year university students revealed a significant association between prolonged internet use and musculoskeletal pain.16 These findings collectively demonstrate the increased prevalence of MSDs among online learners across various countries.\n\nMusculoskeletal injuries can be classified into two main categories: acute and cumulative. Acute injuries result from a direct impact or force, such as a blow or crush, while cumulative injuries develop gradually over time due to repetitive or prolonged postures or activities.17 Online learning, for instance, can contribute to cumulative musculoskeletal injuries. A study in Jordan examining neck pain among online learners using smartphones revealed that 43.9% of students who spent 10-30% of their day engaged in online learning reported neck pain.18 Additionally, a study investigating the causes of musculoskeletal symptoms during lockdown measures revealed that among 319 participants aged 18-60 years who were confined to their homes, the most common reasons for pain were increased phone usage (43.7%), prolonged sitting (41.3%), and lack of physical activity (29.4%). The most prevalent pain locations were the lower back (62.2%), neck (48%), and upper back (35.4%).19 MSDs can cause pain in muscles, tendons, bones, or joints throughout the body and can significantly impact daily life if severe. These disorders can affect individuals of all ages, including children, not just those in the working population.20\n\nMSDs have been extensively studied in various occupations, including athletes (e.g., runners,21 ballet dancers22 and healthcare workers (e.g., dentists,23 nurses24). However, a systematic review and meta-analysis of MSD prevalence among online learners during the COVID-19 pandemic has not yet been conducted. Therefore, this study aimed to systematically review and meta-analyze the prevalence of MSDs among online learners during the COVID-19 pandemic to quantify the magnitude of the problem and inform future research directions.\n\n\nMethods\n\nThis study is reported following the NEW Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines.25 The study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (Registration number: CRD42022335229).\n\nThe search for relevant studies was conducted in electronic databases, including Medline via PubMed, Cochrane Library, Wiley InterScience, SCOPUS, ProQuest, and SciendDirect. The search included studies published between January 2020 and December 2023. Keywords were searched individually and in combination using Boolean operators such as “OR” and “AND.” The following medical subject headings (MeSH) were used: Prevalence, Musculoskeletal, Low back pain, School student, College student, University student, Adolescent, Online Learning, Distance Education, Distance Learning, Online Education, Remote Learning, COVID-19, Computer, and Smartphone (Extended data: Supplementary file 1).\n\nResearch articles meeting the following criteria were included in the study: 1) target population: university students; 2) study context: online learning during the COVID-19 pandemic; 3) research objective: to investigate the prevalence of MSDs among online learners; 4) study design: cross-sectional study; 5) publication date: January 2020 to December 2023; 6) language: English; and 7) publication type: peer-reviewed journal.\n\nExclusion criteria: 1) Nonreporting of prevalence: Research articles that did not report the prevalence of MSDs among online learners were excluded. 2) Inaccessible full-text: Research articles for which the full text could not be accessed were excluded.\n\nTwo independent reviewers (TG and OK) screened the search results, initially assessing potential studies based on titles and abstracts. For studies that appeared to meet the inclusion criteria, the full-text articles were retrieved. Any discrepancies in the screening reports were resolved through consensus with a third reviewer (WJ).\n\nData were extracted from articles that met the inclusion and exclusion criteria for further analysis. The data extracted included author, year, country, sampling frame, study sample size, response rate, period of data collection, tool for MSDs, period for pain, prevalence of symptoms, and duration of use/online.\n\nThe Joanna Briggs Institute (JBI) critical appraisal checklist for prevalence studies was used to evaluate the methodological quality of the included studies.26 This tool assesses studies based on nine questions. A score of 1 is awarded for each “yes” answer, while a score of 0 is given for “no,” “unclear,” or “not applicable” responses. Overall quality scores ≤ 4, 5-7, and ≥ 8 were considered low, moderate, and high, respectively. Two reviewers (TG and OK) independently conducted the quality assessments, and any discrepancies were resolved through discussion.\n\nJamovi 2.4 was used in the meta-analysis. Prevalence estimates for MSDs were calculated using percentages and 95% confidence intervals (CIs). Heterogeneity of the included studies was assessed using Cochran’s Q test with a significance level of 0.05. If there was significant heterogeneity between studies (p value < 0.05), a random effects model was used to estimate the pooled prevalence. For studies with nonsignificant heterogeneity (p value ≥ 0.05), a fixed-effects model was used to calculate the pooled prevalence.\n\nPublication bias was evaluated using funnel plots, Egger’s test, with a significance level of 0.05. If publication bias was detected, efforts were made to identify and report potential sources of bias.\n\n\nResults\n\nThe search yielded 6,016 articles and an additional 32 articles from the reference checking of the collected studies. After checking for duplicates and applying the inclusion and exclusion criteria, 5,350 articles remained. Based on the title and abstract, 60 articles remained. After a thorough review of the full-text articles, 16 articles remained. These were entered into the article assessment and meta-analysis (Figure 1).25\n\nAs shown in Table 1, the characteristics of the study population were as follows: medical students (n = 5),27–31 nursing students (n = 3),31–33 dental students (n = 1),29 pharmacy students (n = 1),29 physical therapy students (n = 1),34 physical education and sports students (n = 1),35 and university students without specified faculty (n = 6).32,36–40 The sample sizes of these studies ranged from 120 to 3,705 participants. The mean age was 22 years (Extended data: Supplementary file 2).\n\n\n\n1. Vernon Mior’s Neck Disability Index (NDI)\n\n2. Roland–Morris Disability\n\nQuestionnaire (for LBP)\n\nAssessment of the research quality by two researchers (TG and OK) revealed that there were six high-quality studies, nine moderate-quality studies, and one low-quality study. The studies investigating musculoskeletal symptoms were conducted in six European countries,34–36,38,39,41 nine Asian countries,29–33,37,40,42 and one South American country (Figure 2).27\n\nA meta-analysis was conducted to determine the prevalence of musculoskeletal symptoms in 9 body regions according to the Standardized Nordic Questionnaire (SNQ).43 Additional body regions were added to align with the prevalence reported in the reviewed articles, including the upper arm and lower arm, for a total of 11 regions. The results are presented for three groups: 1) axial body: neck, upper back, and lower back; 2) upper limbs: shoulder, upper arm, lower arm, elbow, and wrist/hand; and 3) lower limbs: hip/thigh, knee, and ankle/foot.\n\nThe axial body region had the highest prevalence of musculoskeletal symptoms, with the neck (95% CI 36-66%) and lower back (95% CI 42-59%) having an equal prevalence of 51%. The prevalence of hypertension in the upper back was 36% (95% CI 26–47%) (Figure 3). The upper limbs had the highest prevalence of musculoskeletal symptoms, with the shoulder (95% CI 19-44%) and upper arm (95% CI 9-53%) having an equal prevalence of 31%. The lower arm had a prevalence of 28% (95% CI 5-52%). The elbow had the lowest prevalence of musculoskeletal symptoms, at 11% (95% CI 1 - 22%) (Figure 4). The lower limbs had the highest prevalence of injuries, with the hip having a prevalence of 25% (95% CI 16–34%). The prevalence of knee involvement was 18% (95% CI 7-28%), and the prevalence of ankle/foot involvement was 17% (95% CI 11-23%) (Figure 5).\n\nSubgroup analyses were conducted for the axial body (neck, upper back, lower back) to examine the heterogeneity between studies. The heterogeneity was not significant for any of the subgroups (I2 = 99.69-98.20). Egger’s regression test was used to assess publication bias. No evidence of publication bias was found in the meta-analysis of the overall prevalence of pain (p value < 0.001). Similarly, no evidence of publication bias was found for the upper limbs (shoulder, upper arm, elbow, forearm, wrist/hand) (I2 = 99.85-99.17, p value < 0.001) or lower limbs (hip, knee, lower leg) (I2 = 99.43-97.47, p value < 0.001) (Extended data: Supplementary file 3 and Table 2).\n\n\nDiscussion\n\nThis systematic review and meta-analysis aimed to investigate the prevalence of musculoskeletal symptoms in different body regions among individuals engaged in online learning during the COVID-19 pandemic. A total of 16 studies were included, comprising six high-quality studies, nine moderate-quality studies, and one low-quality study. The studies were conducted in six European countries, nine Asian countries, and one South American country. The sample sizes ranged from 120 to 3,705 participants.\n\nThe results of this meta-analysis on the prevalence of musculoskeletal symptoms among individuals engaged in online learning during the COVID-19 pandemic revealed that the most frequently reported pain locations were the neck, lower back, and shoulders. The least frequently reported pain locations were the upper arm, lower arm, and knee. These findings are consistent with previous studies on musculoskeletal injuries among work-from-home employees during the COVID-19 pandemic, which reported prevalence rates of 20.3-76.9% for neck pain, 19.5-74.1% for lower back pain, and 3.0-72.9% for shoulder pain.44 Additionally, a study conducted among students and faculty members of the Faculty of Public Health in Thailand reported a 34% prevalence of upper arm pain during periods of online learning and work from home,45 which is similar to the findings of our meta-analysis.\n\nFor the lower limbs, the results of this study revealed that the most prevalent musculoskeletal symptom was located in the hip, with an overall prevalence of 25% [95% confidence interval (CI) 16–34%]. This finding is consistent with a study conducted among online teachers during the COVID-19 pandemic in Brazil, which reported a prevalence of hip pain of 25%.46 This is likely due to prolonged sitting in the same position during online learning, which can lead to more hip pain than in other areas, such as the knee and ankle/foot, which are more commonly associated with pain in athletes.47\n\nMusculoskeletal pain in students can arise from various factors, including poor posture, prolonged sitting or standing, improper ergonomics,48 stress,40 and lack of physical activity.9 Studies investigating online learning environments suggest a potential association between prolonged screen time and poor posture.28,49 This finding aligns with established research demonstrating that maintaining a hunched posture during prolonged reading, texting, or laptop use can strain muscles and lead to pain in the neck, back, and shoulders.39,50,51 Notably, the current study did not examine the specific risk factors associated with musculoskeletal pain in this online learning population.\n\nStudies conducted prior to the COVID-19 outbreak have established the prevalence of musculoskeletal pain.52 However, the COVID-19 pandemic appears to have significantly increased its prevalence. Reports indicate an increase in the prevalence of musculoskeletal pain, ranging from 15.60% to 64.40%, possibly attributable to increased screen time and reduced physical activity levels associated with lockdowns.53 While respiratory complications are well established in patients with COVID-19, musculoskeletal manifestations are increasingly being recognized.54,55 During the acute phase, patients with COVID-19 may experience fatigue, myalgia, arthralgia, back pain, and chest pain. These symptoms can also persist after contracting COVID-19.56 The specific causes of musculoskeletal involvement in patients with COVID-19, potentially including viral effects, medications, and immobilization, remain under investigation.57,58 Emerging evidence suggests an increased prevalence of musculoskeletal symptoms during COVID-19 infection.\n\nThis systematic review has some limitations that should be considered. First, the causes of musculoskeletal pain resulting from online learning have not yet been adequately studied, as insufficient and diverse research is available. However, there is a clear understanding of the causes of MSDs from online learning, which are primarily related to physical ergonomic factors.59 Second, the study designs included in this review were cross-sectional, which means that the duration of online learning that leads to musculoskeletal pain could not be analyzed. Finally, this study could not analyze ergonomic factors to identify the types of devices involved. Therefore, further research is needed to investigate the duration of online learning that contributes to musculoskeletal pain. This information could be used to develop guidelines for dividing online learning into appropriate periods throughout the day to minimize musculoskeletal injuries among students. Additionally, further research is needed to examine the types of electronic devices that have the greatest impact on musculoskeletal injuries. This information could be used to inform recommendations for device selection and usage to reduce the risk of musculoskeletal injuries among students.\n\n\nConclusion\n\nThe shift to online learning is another factor that may affect musculoskeletal pain in students. However, there is insufficient evidence to determine the specific duration of online learning per day that begins to impact the presence of musculoskeletal injuries in learners.\n\n\n\n• Setting up an ergonomic workspace at home (chair height, monitor position, etc.) and maintaining good posture while using electronic devices.\n\n• Encourage teachers to incorporate short physical activity breaks into online lessons. This could involve simple stretches, standing desks, or short bursts of jumping jacks.\n\n• Develop resources or guidelines for students on recognizing early signs of discomfort and appropriate self-care strategies (e.g., applying heat/ice, modifying postures, etc.).\n\n\nAuthor contributions\n\nTG, OK and WJ designed the study. TG and OK acquired the data. TG and OK analyzed and interpreted the data. TG and OK wrote the initial draft. All authors critically reviewed and approved the final manuscript. All authors had final responsibility for the decision to submit for publication.", "appendix": "Data availability statement\n\nNo data are associated with this article.\n\nMendeley Data: Musculoskeletal symptoms from online learning: systematic review and meta-analysis, https://data.mendeley.com/datasets/jhggs6tbw4/2. 60\n\nThis project contains the following underlying data:\n\nSupplementary file 1: Search strategies.\n\nSupplementary file 2: Study prevalence.\n\nSupplementary file 3: Funnel plot.\n\nPRISMA_2020_checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\norganization wh: WHO coronavirus (COVID-19) dashboard. World health organization; 2022 [updated 31 May 2022]. Reference Source\n\nOnyeaka H, Anumudu CK, Al-Sharify ZT, et al.: COVID-19 pandemic: a review of the global lockdown and its far-reaching effects. Sci. Prog. 2021; 104(2): 00368504211019854. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDepartment of Disease Control: The Department of Disease Control reveals guidelines for people living in the most controlled areas. As for provinces with no reported cases Please take strict measures.2021 [updated 4 January 2021; cited 2024 14 April]. Reference Source\n\nOffice of the Basic Education Commission: Guidelines for organizing distance learning during the COVID-19 outbreak situation.2020 20 May [cited 2024 14 April]:[2 p.]. Reference Source\n\nDepartment of Mental Health: Thai Health Promotion Foundation brainstorms with partners to find a solution to the problem of stressed children studying online.2021 [cited 2024 14 April]. Reference Source\n\nBull FC, Al-Ansari SS, Biddle S, et al.: World Health Organization 2020 guidelines on physical activity and sedentary behaviour. Br. J. Sports Med. 2020; 54(24): 1451–62. Epub 2020/11/27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWibowo DMRRA: Changes in physical activity among university students in Indonesia from before to during the COVID-19 pandemic: a retrospective cohort study. Journal of Population and Social Studies (JPSS). 2022; 30: 134.\n\nSchmidt SCE, Anedda B, Burchartz A, et al.: Physical activity and screen time of children and adolescents before and during the COVID-19 lockdown in Germany: a natural experiment. Sci. Rep. 2020; 10(1): 21780. Epub 20201211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoggio F, Trovato B, Ravalli S, et al.: One year of COVID-19 pandemic in Italy: effect of sedentary behavior on physical activity levels and musculoskeletal pain among university students. Int. J. Environ. Res. Public Health. 2021; 18(16). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong Y, Jiang P, Jin X, et al.: Association between long-term static postures exposure and musculoskeletal disorders among university employees: A viewpoint of inflammatory pathways. Front. Public Health. 2022; 10: 1055374. Epub 2022/12/20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaringada T, Sony M: Demonstration of the relationship between MSD and online learning during the COVID-19 pandemic. Journal of Applied Research in Higher Education. 2021; 14: 200–222. Publisher Full Text\n\nJafari Nodoushan Abbasali BG, Fatemesadat MN: Effect of COVID-19 virus on Prevalence of Musculoskeletal Disorders of Faculty Members of Yazd University. JOURNAL OF ERGONOMICS. 2022; 8(3): 1–12.\n\nReuter PR, Fichthorn KR: Prevalence of generalized joint hypermobility, musculoskeletal injuries, and chronic musculoskeletal pain among American university students. PeerJ. 2019; 7: e7625. Epub 20190911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHupert N, Amick BC, Fossel AH, et al.: Upper extremity musculoskeletal symptoms and functional impairment associated with computer use among college students. Work. 2004; 23(2): 85–93. PubMed Abstract\n\nOgunlana MO, Govender P, Oyewole OO: Prevalence and patterns of musculoskeletal pain among undergraduate students of occupational therapy and physiotherapy in a South African university. Hong Kong Physiother. J. 2021; 41(1): 35–43. Epub 20210118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang G, Cao J, Li Y, et al.: Association between internet addiction and the risk of musculoskeletal pain in Chinese College Freshmen – a cross-sectional study. Front. Psychol. 2019; 10: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGraveling R: Ergonomics and Musculoskeletal Disorders (MSDs) in the Workplace: A Forensic and Epidemiological Analysis. CRC Press; 2018.\n\nGaowgzeh RA, Mustafa A: The use of smartphones in distance learning/e-learning/online classes and its impact to neck pain in the light of the Covid-19 pandemic.2020.\n\nAhmed S, Akter R, Islam MJ, et al.: Impact of lockdown on musculoskeletal health due to COVID-19 outbreak in Bangladesh: A cross sectional survey study. Heliyon. 2021; 7(6): e07335. Epub 20210617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Academies of Sciences E, and Medicine; Health and Medicine Division; Board on Health Care Services; Committee on Identifying Disabling Medical Conditions Likely to Improve with Treatment: Selected Health Conditions and Likelihood of Improvement with Treatment. Washington (DC): National Academies Press (US); 2020 [cited 2024 3 May]. Reference Source\n\nKakouris N, Yener N, Fong DTP: A systematic review of running-related musculoskeletal injuries in runners. J. Sport Health Sci. 2021; 10(5): 513–22. Epub 20210420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith TO, Davies L, de Medici A , et al.: Prevalence and profile of musculoskeletal injuries in ballet dancers: a systematic review and meta-analysis. Phys. Ther. Sport. 2016; 19: 50–6. Epub 20160105. PubMed Abstract \| Publisher Full Text\n\nHayes M, Cockrell D, Smith DR: A systematic review of musculoskeletal disorders among dental professionals. Int. J. Dent. Hyg. 2009; 7(3): 159–165. PubMed Abstract \| Publisher Full Text\n\nBernal D, Campos-Serna J, Tobias A, et al.: Work-related psychosocial risk factors and musculoskeletal disorders in hospital nurses and nursing aides: a systematic review and meta-analysis. Int. J. Nurs. Stud. 2015; 52(2): 635–48. Epub 20141115. PubMed Abstract \| Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEpstein S, Sparer EH, Tran BN, et al.: Prevalence of work-related musculoskeletal disorders among surgeons and interventionalists: a systematic review and meta-analysis. JAMA Surg. 2018; 153(2): e174947. Epub 20180221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomes I, Mitleton V, Fiorin L, et al.: Musculoskeletal pain in medical students subject to remote teaching during the COVID-19 pandemic. Journal of Biosciences and Medicines. 2021; 09: 19–92. Publisher Full Text\n\nSalameh MA, Boyajian SD, Odeh HN, et al.: Increased incidence of musculoskeletal pain in medical students during distance learning necessitated by the COVID-19 pandemic. Clin. Anat. 2022; 35(4): 529–536. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlmhdawi KA, Alazrai A, Obeidat D, et al.: Healthcare students’ mental and physical well-being during the COVID-19 lockdown and distance learning. Work. 2021; 70(1): 3–10. PubMed Abstract \| Publisher Full Text\n\nSamarah OQ, Maden HA, Sanwar BO, et al.: Musculoskeletal pain among medical students at two Jordanian universities. J. Back Musculoskelet. Rehabil. 2023; 36(2): 429–436. PubMed Abstract \| Publisher Full Text\n\nMahmoud NA, Abu Raddaha AH, Zaghamir DE: Impact of digital device use on neck and low back pain intensity among nursing students at a Saudi Government University: a cross-sectional study. Healthcare (Switzerland). 2022; 10(12). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSirajudeen MS, Alzhrani M, Alanazi A, et al.: Prevalence of upper limb musculoskeletal disorders and their association with smartphone addiction and smartphone usage among university students in the Kingdom of Saudi Arabia during the COVID-19 pandemic—a cross-sectional study. Healthcare. 2022; 10: 2373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDireksunthorn T, Polpanadham P, Summart U, et al.: Association of electronic learning devices and online learning properties with work-related musculoskeletal disorders (WMSDs): A cross-sectional study among Thai undergraduate students. PLoS One. 2023; 18(10 October): e0291597. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerlito R, Panebianco P, Rizzo V, et al.: The onset of musculoskeletal pain in the COVID-19 Era: a survey of physiotherapy students in Sicily. Journal of Functional Morphology and Kinesiology. 2023; 8(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilișteanu SC, Totan M, Antonescu OR, et al.: The impact of COVID-19 on behavior and physical and mental health of Romanian College Students. Medicina. 2022; 58(2): 246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElghomati A, Korhan O, Sekeroglu B, et al.: Short-term impacts of mobile touch-screen device use on musculoskeletal disorders during Covid-19 pandemic: risk assessment modelling and verification. South African Journal of Industrial Engineering. 2022; 32(2): 62–77. PubMed Abstract \| Publisher Full Text\n\nKaringada KT, Sony M: Demonstration of the relationship between MSD and online learning during the COVID-19 pandemic. Journal of Applied Research in Higher Education. 2022; 14(1): 200–222. Publisher Full Text\n\nRoggio F, Trovato B, Ravalli S, et al.: One year of COVID-19 pandemic in Italy: effect of sedentary behavior on physical activity levels and musculoskeletal pain among university students. Int. J. Environ. Res. Public Health. 2021; 18(16): 8680. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanc M, Jozwiak Z, Jankowska A, et al.: Ergonomics of E-Learning Workstations and the Prevalence of Musculoskeletal Disorders-Study among University Students. Int. J. Environ. Res. Public Health. 2023; 20(4). Epub 20230214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarithasan D, Singh DKA, Abd Razak NAB, et al.: Personal, Academic Stressors and Environmental Factors Contributing to Musculoskeletal Pain among Undergraduates Due to Online Learning: A Mixed Method Study with Data Integration. Int. J. Environ. Res. Public Health. 2022; 19(21). Epub 20221104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeirós-Rodríguez R, Rodríguez-Nogueira Ó, Pinto-Carral A, et al.: Musculoskeletal Pain and Non-Classroom Teaching in Times of the COVID-19 Pandemic: Analysis of the Impact on Students from Two Spanish Universities. J. Clin. Med. 2020; 9(12): 4053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahmoud H, El-Bidawy RA, Daadour M, et al.: Correlation between low back pain among medical students and prolonged unhealthy sitting with e-learning during COVID-19 pandemic. JOURNAL OF HEALTHCARE SCIENCES. 2021; 1(7): 6.\n\nCrawford JO: The Nordic Musculoskeletal Questionnaire. Occup. Med. 2007; 57(4): 300–301. Publisher Full Text\n\nAlzhrani A, Cook M, Johnstone K, et al.: Handheld Mobile Devices—How Do We Use Them at Work? A University Case Study. Musculoskeletal Disorders. 2019; 820: 126–137. Publisher Full Text\n\nTongdi N, Inwan C, Proytong S, et al.: Muscle pain and risk factors of muscle pain during the online learning and working from home among the students and staffs of Faculty of Public Health, Thammasat University, Lampang Campus. Thai Journal of Ergonomics. 2021; 4(1): 2021.\n\nMatias NMS, Bezerra LÂ, Nascimento SEA, et al.: Correlation between musculoskeletal pain and stress levels in teachers during the remote teaching period of the COVID-19 pandemic. Fisioterapia em Movimento. 2022; 35: 35. Publisher Full Text\n\nGoes RA, Lopes LR, Cossich VRA, et al.: Musculoskeletal injuries in athletes from five modalities: a cross-sectional study. BMC Musculoskelet. Disord. 2020; 21(1): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalshani W, Weerakoon H, Weerakoon K: Ergonomics in online education of medical undergraduates: A challenge to post-COVID transformation in educational activities. Work. 2023; 76(1): 21–31. Epub 2023/05/30. PubMed Abstract \| Publisher Full Text\n\nSiripoon P, Wongchalee N, Wittayapun Y, et al.: Prevalence and factors associated with musculoskeletal pain during online learning among nursing students in a university. Journal of Nursing Science and Health. 2022; 46(1): 21–33.\n\nFan LJ, Liu S, Jin T, et al.: Ergonomic risk factors and work-related musculoskeletal disorders in clinical physiotherapy. Front. Public Health. 2022; 10: 1083609. Epub 2023/01/07. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzczygieł E, Zielonka K, Mętel S, et al.: Musculo-skeletal and pulmonary effects of sitting position - a systematic review. Ann. Agric. Environ. Med. 2017; 24(1): 8–12. Epub 2017/04/06. PubMed Abstract \| Publisher Full Text\n\nGran JT: The epidemiology of chronic generalized musculoskeletal pain. Best Pract. Res. Clin. Rheumatol. 2003; 17(4): 547–61. Epub 2003/07/10. PubMed Abstract \| Publisher Full Text\n\nSaumya P, Prajapati AP: Prevalence of musculoskeletal disorder among college students in times of COVID-19 pandemic - an observational study. International Journal of Health Sciences and Research. 2021; 11(10): 214–219. Publisher Full Text\n\nEvcik D: Musculoskeletal involvement: COVID-19 and post COVID 19. Turk J Phys Med Rehabil. 2023; 1(1): 1–7. Epub 2023/05/18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nda Silva LNM , Filho AGO, Guimarães JB: Musculoskeletal manifestations of COVID-19. Skeletal Radiol. 2023. PubMed Abstract \| Publisher Full Text\n\nAlkodaymi MS, Omrani OA, Fawzy NA, et al.: Prevalence of post-acute COVID-19 syndrome symptoms at different follow-up periods: a systematic review and meta-analysis. Clin. Microbiol. Infect. 2022; 28(5): 657–666. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOmar IM, Weaver JS, Samet JD, et al.: Musculoskeletal manifestations of COVID-19: currently described clinical symptoms and multimodality imaging findings. RadioGraphics. 2022; 42(5): 1415–1432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDisser NP, De Micheli AJ, Schonk MM, et al.: Musculoskeletal consequences of COVID-19. JBJS. 2020; 102(14): 1197–1204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPunnett L, Wegman DH: Work-related musculoskeletal disorders: the epidemiologic evidence and the debate. J. Electromyogr. Kinesiol. 2004; 14(1): 13–23. Epub 2004/02/05. PubMed Abstract \| Publisher Full Text\n\nGotum T, Keeratisiroj O, Jariya W: Musculoskeletal symptoms from online learning: systematic review and meta-analysis. Mendeley Data. 2024; V2. Publisher Full Text" }	[ { "id": "321523", "date": "12 Sep 2024", "name": "Anna Zalewska", "expertise": [ "Reviewer Expertise Health Sciences" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this paper. This is a timely issue to explore. The aim of this study was to assess the prevalence of musculoskeletal symptoms in online students. The manuscript meets all the requirements for a systematic review. I have two minor comments for the authors. - Authors should also remember to exclude so-called grey literature from their research. - Research articles usually do not use the word „our” and regularly use passive verbs.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] }, { "id": "331325", "date": "31 Oct 2024", "name": "Mati Pääsuke", "expertise": [ "Reviewer Expertise musculoskeletal 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\nGeneral Comments This is an interesting systematic review and meta-analysis, assessing the prevalence of musculoskeletal disorders (MSDs) in different body regions among online learners during the COVID-19 pandemic. A total 16 full-text articles from different electronic databases were included in this meta-analysis. The participants were University students with mean age of 22 years from 6 European countries, 9 Asian countries and 1 South American country. This meta-analysis suggested, that the areas with the highest prevalence of MSDs in online learners were the neck (51%), lower back (51%) and shoulder (36%). The Authors concluded that the shift to online learning during the COVID-19 pandemic has emerged as a potential factor influencing musculoskeletal pain in students.\nThe manuscript is generally well written. However, the design of this paper should be slightly improved before getting approved for indexing.\nIn my opinion, it is obligatory to present in Abstract and Results (Study selection and characteristics) information about age range and sex (% female) of the participants for better understanding the measured subjects.\n\nThis study has several additional limiting factors: (1) Cultural (participants from 16 countries, and from Europe, Asia and South America) differences may have possibly influenced the individuals’ understanding and interpretation of the musculoskeletal pain. (2) The gender-related differences in MSDs of the participants was not analyzed in this meta-analyses. (3) Habitual physical activity of the participants was not analyzed in this study. All these limitations should be presented at the end of the Discussion.\n\nIn conclusion, I recommend to accept this manuscript for indexing in bibliographic databases given that the authors address all the minor corrections.\nSpecific Comments Abstract Page 1. Please add the information about age range and sex (% female) of the participants.\nResults Page 5, Study selection characteristics. Please add information about age range and sex (%female) of the participants. Discussion Page 12. Please add more limiting factors (see General Comments)\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-790
https://f1000research.com/articles/12-1492/v1	22 Nov 23	{ "type": "Case Report", "title": "Case Report: Disseminated hydatid cyst: Unusual presentation and therapeutic challenges", "authors": [ "Anas Mohamed Babiker", "Amro Abdelrahman", "Asmaa Abdalkarim", "Gufran Algaly", "Amin Sanosi", "Elhassan Mohamed Abdalla", "Hany A Zaki", "Moayad Elgassim", "Muhnad Abdeen", "Mohamed Elgassim", "Amro Abdelrahman", "Asmaa Abdalkarim", "Gufran Algaly", "Amin Sanosi", "Elhassan Mohamed Abdalla", "Hany A Zaki", "Moayad Elgassim", "Muhnad Abdeen", "Mohamed Elgassim" ], "abstract": "Introduction: Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a parasitic disease prevalent in endemic regions. CE frequently leads to the formation of hydatid cysts in various organs, with the liver being the most commonly affected site. Involvement of the spleen has been rarely reported in the literature. Managing disseminated hydatid cyst disease presents significant diagnostic and therapeutic challenges.\nCase presentation: A 40-year-old female with a past medical history of hypothyroidism presented with sudden onset shortness of breath, dry cough, and chest pain for 3 days. She had a recent travel history to Egypt. Physical examination revealed mild right upper quadrant tenderness. Laboratory findings showed elevated white blood cell count with eosinophilia and increased inflammatory markers. Chest X-ray and pan-computed tomography (Pan-CT) scans identified multiple cystic lesions in the lung, liver and spleen. Serological tests confirmed the presence of anti-Echinococcus antibodies, leading to a diagnosis of disseminated hydatid cyst disease. The patient was managed medically and surgically by a multidisciplinary team.\nConclusion: Disseminated hydatid cyst disease, though rare, presents complex diagnostic and management challenges. Timely recognition, supported by clinical, radiological, and serological assessments, is essential. Surgical intervention should be considered in a patient when multiple extrahepatic cysts are present, and rupture is evident, as this approach can significantly reduce patient morbidity and mitigate life-threatening complications.", "keywords": [ "Disseminated hydatid cyst", "Cystic echinococcosis", "Spleen cyst", "Lung cyst", "Liver cyst", "Case report" ], "content": "Introduction\n\nThe hydatid disease, cystic echinococcosis (CE), is a parasitic disease caused by Echinococcus granulosus sensu lato. In endemic countries, CE shows an average incidence of 200 per 100,000 populations with a mortality range of 2–4% when appropriate treatment is provided promptly.1 The larval form of the deadly tapeworm may lodge itself in various different sites and organs to form fluid-filled sacs known as hydatid cysts. The disease may manifest through dysfunction of the organs where the cysts form. With the liver being the most vulnerable, it alone accounts for nearly 70% of cases, while the lungs account for 20% of deposits. Other organs may also be involved in other rarer cases, including the kidneys, muscles, brain, and spleen.2 In this case, we are reporting a 40-year-old female patient that presented to the hospital with sudden onset shortness of breath, a dry cough, and chest pain for only 3 days. Radiological reports showed hydatid cyst involvement of the liver, the lungs, and the spleen, with positive antibodies on serological studies. She was diagnosed as a case of disseminated hydatid cyst and managed by a multidisciplinary team.\n\n\nCase presentation\n\nA 40-year-old Egyptian female, previously diagnosed with hypothyroidism and currently taking thyroxine, presented to the hospital with sudden onset shortness of breath, a dry cough, and chest pain for 3 days. The patient denied any orthopnea or paroxysmal nocturnal dyspnea. The review of other systems was unremarkable. Apart from thyroxine, the patient was not taking any other medications. She had traveled to Egypt two months before her presentation. In the emergency department, her vital signs were within normal limits. Her temperature was 36.7°C, her blood pressure was 136/72 mmHg, and she had a heart rate of 86 beats per minute, all while maintaining normal saturation on room air. On physical examination, she was in distress; auscultation of her chest was clear with no crackles or rhonchi. An abdominal examination revealed only minor tenderness in the right upper quadrant. Examination of other systems was unremarkable. Results of her lab investigations revealed a high white blood cell (WBC) count of 12,000 (normal range=4,000–11,000) with prominent eosinophils at 3.5 (normal range= 0–0.5) and elevated C-reactive protein level of 25 (normal range= 0–5).\n\nA chest X-ray showed multiple well-defined radiopaque rounded densities (Cannon balls) scattered throughout both lungs and more on the right side (Figure 1).\n\nMultiple well-defined radiopaque rounded densities (Cannon balls) scattered throughout both lungs.\n\nThe radiologist recommended a pan-computed tomography (PAN CT) scan, which revealed multiple large, well-circumscribed bilateral pulmonary cystic lesions representing hydatid cysts. The largest cyst measured 6 cm in the anterior segment of the right upper lobe. In the posterior basal region of the left lobe, a ruptured hydatid cyst with surrounding consolidation was discovered. Furthermore, multiple enlarged, variable-size subcapsular cystic lesions with multiple curvilinear hyperattenuating structures were seen in both lobes of the liver. The largest is a 5-cm-segment V/VI image with no postcontrast enhancement. A 7.5-cm-enhancing cystic lesion in the upper pole of the spleen was also seen (Figure 2).\n\n(A) well-circumscribed bilateral pulmonary cystic lesions representing hydatid cysts. The largest cyst measured 6 cm in the anterior segment of the right upper lobe.\n\n(B) Bilateral multiple cysts.\n\n(C) Ruptured hydatid cyst with surrounding consolidation in the posterior basal region of the left lobe.\n\n(D) Multiple enlarged, variable-size subcapsular cystic lesions with multiple curvilinear hyperattenuating structures in both lobes of the liver. The largest is a 5-cm-segment V/VI image with no postcontrast enhancement. A 7.5-cm-enhancing cystic lesion in the upper pole of the spleen.\n\nThe image findings supported the diagnosis of disseminated hydatid disease, and serological tests for echinococcus antibodies were positive. Urgent consultations were obtained with both the infectious disease and thoracic surgery teams. The infectious disease (ID) team recommended 400 mg of albendazole orally twice daily with follow-up until resolution. The thoracic surgery team advised her to return to the clinic after two weeks of stabilization for elective surgery and to consult with hepatobiliary surgeons for liver and spleen cysts. The plan was to surgically remove the lung cysts first, followed by the liver and spleen cysts later, along with albendazole, which was prescribed by the ID team. After three days of stabilization and resolution of symptoms, the patient was discharged with an outpatient follow-up with the infectious disease, thoracic surgery, and hepatobiliary surgery teams. One week after being discharged, the patient returned with a severe dry cough and chest pain. A new CT scan of the chest revealed that a new cyst had ruptured in the right upper lobe of the lung (Figure 3).\n\nRuptured cyst in the right upper lobe of the lung.\n\nThe patient underwent an emergency thoracotomy with multiple cystectomies, and histopathological examination of the surgical specimen revealed findings consistent with hydatid cysts. Six months later, a follow-up CT scan of the chest showed cyst resolution. The patient had a successful laparoscopic deroofing and endocystectomy of hepatic and splenic cysts one month later, and the pathological analysis of the surgical specimen from hepatic and splenic tissues showed findings consistent with a hydatid cyst.\n\n\nDiscussion\n\nHydatid cysts are categorized as zoonotic infections that primarily affect dogs, which serve as definitive hosts when they consume contaminated sheep, goats, or various animal tissues. A single cyst can contain numerous protoscolices, each capable of maturing into an adult tapeworm upon ingestion by the definitive host. Animals acquire this infection through the consumption of contaminated food. In humans, accidental intermediate hosts become infected through the consumption of infected food or water, contact with contaminated soil, or direct contact with definitive hosts. The larval stage enters the bloodstream and infiltrates various organs in the body, particularly those with high blood supplies.3,4 Echinococcus granulosus cysts can inhabit any anatomical site. The clinical manifestation depends on several factors, including the affected organ, specific location within the organ, stage of cyst development, and vitality of the cyst contents. In most cases, these cysts remain silent and are often discovered incidentally during routine imaging or postmortem examinations. However symptoms may arise as a result of cyst enlargement, causing pressure effect on adjacent structures, potential infection, or the rupture of cyst contents into adjacent body cavities.5 After infection, the parasitic infection can disseminate to different organs via the lymphatic or venous system, with the liver being the most frequently affected organ initially. Appropriate treatment reduces the risk of further spread.6 Our patient presented with a cough, shortness of breath, and chest pain due to a ruptured cyst. In addition to that, his physical examination showed right upper abdominal tenderness.\n\nThe diagnosis of a hydatid cyst can be established through combination of clinical assessment, laboratory and serological studies, radiological evidence, and histopathological analysis. In addition to identifying elevated inflammatory markers and a high eosinophil count in the bloodstream, serological tests of our patient also revealed the presence of anti-Echinococcus antibodies. A previous research study has proposed that eosinophilia in individuals afflicted with echinococcosis lacks specificity and has advocated for the inclusion of serological studies in the diagnostic process. These serological tests exhibit a sensitivity of 80% to 100% for hepatic echinococcosis, 50% to 56% for pulmonary echinococcosis, and 25% to 50% for detecting involvement of other organs.7 Generally, the classic radiological finding of a hydatid cyst of the lung is a well-defined oval to rounded opacity ranging from 1 to 20 cm in size. Usually, lung cysts are located in the lower lobes (60% of cases) and can be multiple and bilateral.8 In cases of hepatic involvement, the most common affected portion is the right portion of the liver. The third most common site of involvement, following the liver and the lung, is the spleen. Splenic hydatid cysts are relatively rare and usually solitary, and their imaging characteristics are similar to those of hepatic hydatid cysts.9 A previous study also reported a case of disseminated echinococcosis involving the lung, liver, and spleen with predominantly abdominal symptoms and pleural effusion on radiological studies.10 In our case, a chest X-ray was ordered, and it showed various rounded radiopaque densities, which prompted further evaluation by a pan-computed tomography (pan-CT) scan. The pan-CT results showed multiple cystic lesions in both lungs. The largest cyst was 6 cm in size and located on the left lobe, and a ruptured cyst on the right lobe was also noted. In addition to pulmonary cysts, multiple cystic lesions involving both lobes of the liver and spleen were also detected, supporting the diagnosis of disseminated echinococcosis.\n\nThe management of hydatid cysts is both medical and surgical. The indications for surgical intervention are a large cyst size of 10 cm, concomitant extrahepatic involvement, and an infected cyst.11 There are two types of surgical intervention: radical (pericystectomy) and conservative (partial cystectomy); the surgery type is usually determined based on the cyst’s size and the presence of cysts outside the liver. In a systematic review, researchers compared the results of radical and conservative surgery with and without a course of chemotherapy postoperatively. The result of radical surgery followed by a course of antiparasitic medication has shown a better outcome in terms of length of hospital stay and post-surgical complications compared to conservative surgery. However, the time of radical surgery was longer with a larger amount of blood loss, and additionally, use of antiparasitic medication after surgery showed a better outcome with reduced recurrence regardless of the type of surgery.12 Albendazole is the standard medication for hydatid cysts, as recommended by the World Health Organization (WHO). It is prescribed both before and after surgery to reduce cyst size, prevent relapses, sterilize the cyst, and mitigate post-surgical complications.13 Our patient started first on medical treatment and planned for elective surgery by the cardiothoracic team, but because she developed symptoms a week after resuming medication after the rupture of an additional cyst, she underwent an urgent right thoracostomy with cystectomy, followed by histopathological analysis of the surgical specimen, which showed findings suggestive of a hydatid cyst. The patient was discharged on albendazole oral tablets. On her 6-month follow-up, a CT scan of the lungs showed evidence of resolution of the hydatid cyst. One month later, elective laparoscopic deroofing and endocystectomy for hepatic and splenic cysts were done, and the surgical specimen was investigated in the histopathology lab. The results showed findings consistent with a hydatid cyst.\n\n\nConclusion\n\nPrompt suspicion and diagnosis of a ruptured hydatid cyst are vital, particularly in patients with respiratory symptoms and radiographic evidence of cystic lesions, especially in those originating from endemic regions. Early surgical intervention is strongly advised when multiple extrahepatic cysts are present, and rupture is evident, as this approach can significantly reduce patient morbidity and mitigate life-threatening complications.\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: CARE checklist for “Case report: Disseminated hydatid cyst: Unusal presentation and therapeutic challenges.” https://doi.org/10.5281/zenodo.8398651. 14\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nOpen Access funding provided by Qatar National Library.\n\n\nReferences\n\nSchweiger A, Ammann RW, Candinas D, et al.: Human alveolar echinococcosis after fox population increase, Switzerland. Emerg. Infect. Dis. 2007; 13(6): 878–882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWen H, Vuitton L, Tuxun T, et al.: Echinococcosis: Advances in the 21st Century. Clin. Microbiol. Rev. 2019; 32(2): e00075–e00018. Published 2019 Feb 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlkhotani A, Butt B, Khalid M, et al.: Peripontomedullary hydatid cyst: Case report and literature review. Int. J. Surg. Case Rep. 2019; 55: 23–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJadoon SK, Khan RMI, Jadoon AK, et al.: Disseminated hydatid cyst disease; a rare presentation in a tertiary care hospital of Azad Jammu Kashmir. Int. J. Surg. Case Rep. 2022; 98: 107578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhuroo MS: Hydatid disease: current status and recent advances. Ann. Saudi Med. 2002; 22(1-2): 56–64. PubMed Abstract \| Publisher Full Text\n\nShabbir MU, Ahmed A, Shaukat F, et al.: Disseminated Hydatid Disease in a Child Involving Multiple Organ Systems: A Case Report. Cureus. 2020; 12(1): e6564. Published 2020 Jan 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Comite G, Dognini G, Gaiera G, et al.: Acute echinococcosis: a case report. J. Clin. Microbiol. 2000; 38(12): 4679–4680. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalikian JP, Mudarris FF: Hydatid disease of the lungs. A roentgenologic study of 50 cases. Am. J. Roentgenol. Radium Therapy, Nucl. Med. 1974; 122(4): 692–707. Publisher Full Text\n\nPedrosa I, Saíz A, Arrazola J, et al.: Hydatid disease: radiologic and pathologic features and complications. Radiographics. 2000; 20(3): 795–817. Publisher Full Text\n\nKalita JM, Naveenraj P, Jain V, et al.: Cystic Echinococcosis of Liver and Spleen Communicating to the Lung: A Rare Case. J Lab Physicians. 2022; 14(3): 351–354. Published 2022 Feb 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoshi U, Subedi R, Jayswal A, et al.: Clinical Characteristics and Management of the Hydatid Cyst of the Liver: A Study from a Tertiary Care Center in Nepal. J. Parasitol. Res. 2020; 2020: 8867744. Published 2020 Sep 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaimakhanov Z, Kaniyev S, Serikuly E, et al.: Radical versus conservative surgical management for liver hydatid cysts: A single-center prospective cohort study. JGH Open. 2021; 5(10): 1179–1182. Published 2021 Aug 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVelasco-Tirado V, Alonso-Sardón M, Lopez-Bernus A, et al.: Medical treatment of cystic echinococcosis: systematic review and meta-analysis. BMC Infect. Dis. 2018; 18(1): 306. Published 2018 Jul 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdelrahman A: Case report: Disseminated hydatid cyst: Unusal presentation and therapeutic challenges. Zenodo. 2023. Publisher Full Text" }	[ { "id": "234514", "date": "13 Feb 2024", "name": "Sultan Abdulwadoud Alshoabi", "expertise": [ "Reviewer Expertise Radiology", "Medical 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\nAt the end of the introduction, the authors should write the purpose of this case report and should explain the importance, what is new in this case, and what they want to emphasize by this case report. In case presentation, the authors should write the events as a scenario by periods of time. They should give more details about radiological examinations because radiological examinations are the the clue for diagnosis of hydatid cysts, and usually pathognomonic for hydatid cyst. The authors should add CT image of chest with mediastinal window to show the contents of the fluid cysts. It is better to add abdominal ultrasound images for liver and splenic cysts if available. They should give wider area for the radiological methods of diagnosis of hydatid cyst. In discussion, the authors should compare their findings with previous similar results either in radiological findings or in surgical intervention and outcomes. Other minor corrections such as adding units of WBCs and others are needed.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [ { "c_id": "11332", "date": "04 Apr 2024", "name": "Amro Abdelrahman", "role": "Author Response", "response": "Dear reviewer, Thanks for feedback. At the end of the introduction, the authors should write the purpose of this case report and should explain the importance, what is new in this case, and what they want to emphasize by this case report. We mentioned this in the end of introduction to emphasize on hydatid cyst involvement in liver, lung and spleen and the importance of multidisciplinary team \"In this case, we are reporting a 40-year-old female patient that presented to the hospital with sudden onset shortness of breath, a dry cough, and chest pain for only 3 days. Radiological reports showed hydatid cyst involvement of the liver, the lungs, and the spleen, with positive antibodies on serological studies. She was diagnosed as a case of disseminated hydatid cyst and managed by a multidisciplinary team.\" In case presentation, the authors should write the events as a scenario by periods of time. They should give more details about radiological examinations because radiological examinations are the the clue for diagnosis of hydatid cysts, and usually pathognomonic for hydatid cyst. The authors should add CT image of chest with mediastinal window to show the contents of the fluid cysts. It is better to add abdominal ultrasound images for liver and splenic cysts if available. They should give wider area for the radiological methods of diagnosis of hydatid cyst. We think our case was written as periodic events including initial presentation at ED, 1 week post discharge presentation and 6-month follow up post surgery. We will add mediastinal image to emphasize on radiological assessment. In discussion, the authors should compare their findings with previous similar results either in radiological findings or in surgical intervention and outcomes. We made comparison just in a different way. In 1st and 2nd paragraph we present findings of literature and at the end of the paragraphs we present our findings. Other minor corrections such as adding units of WBCs and others are needed. Units will be added." } ] }, { "id": "234518", "date": "13 Feb 2024", "name": "Gian Luca D'Alessandro", "expertise": [ "Reviewer Expertise Infectious and tropical diseases department", "with expertise in clinical management of patients affected by cystic echinococcosis (CE)." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case reported by A. M. Babiker et al. the patient, an Egyptian woman aged 40 y.o., sought medical attention for respiratory symptoms and multiple parenchymal cystic lesions: 3 in the right lung, 1 in the left lung, at least 3 in the liver and 1 in the spleen. Respiratory symptoms (shortness of breath, cough and chest pain), radiological picture, eosinophilia and positive E. granulosus serology suggested the diagnosis of multi-organ CE complicated by rupture of the left lung cyst. Although no standardized definition of disseminated hydatidosis exists, in our opinion the term should be limited to secondary echinococcosis, i.e. the dissemination of cyst following spillage and seeding of protoscoleces after leakage or rupture, spontaneous or otherwise typically in the peritoneal or pleural cavities. By this definition, this patient would have multi-organ echinococcosis instead, because the cysts are all intra-parenchymal. In lung CE the risk of cyst rupture is higher than in the liver with potential complications such as bronchial and pleural 1. The first chest CT of the patient showed the rupture of one lung cyst into the pleural space with pericystic consolidation. Complicated lung hydatid disease can be dangerous, even deadly (anaphylaxis, asphyxia) and, for this reason, early treatment is essential. The authors correctly based their hypothesis on clinical picture, imaging findings and serology. However, serology only has a confirmatory role and a negative serology does not rule out echinococcosis. Indeed, CE diagnosis is mainly made through imaging. The positive serology can be explained by the presence of multiple cysts with rupture, factors increasing serology sensitivity in the lung. We are interested in knowing which type of serologic test has been used. After diagnosis, the patient was treated with albendazole (ABZ) and the thoracic surgical team suggested to defer surgery to completion of two weeks of medical therapy. Therefore, the woman was discharged and a week later presented again to the E.D. with the rupture of one contralateral lung cyst. We argue that the patient should have been operated immediately at first presentation, without delay. Cysto-pleural fistula poses risk of pleural dissemination and disease relapse and would have required immediate thoracoscopic pleural toilette with protoscolicidal agents (hypertonic solution, iodopovidone), under ABZ prophylaxis (starting the day before the procedure). As second step, the intact right lung cysts could be removed (immediately after stabilization, i.e. one week later). The use of ABZ in the lung is controversial: in uncomplicated lung CE, starting ABZ is not urgent since the risk of treatment-induced cyst rupture (cyst membrane detachment and opening of cysto-bronchial fistula), while it is indicated as peri-operative prophylaxis 1. In the present case, the patient had complicated disease with pleural fistula and required ABZ to prevent the larval spillage (secondary echinococcosis), but in so doing the other lung uncomplicated cysts were damaged by medical therapy and ruptured, requiring emergency surgery. We guess that a close monitoring of the patient without interrupting hospitalization would have been more beneficial for such complications. In the conclusion of their report, the colleagues state that “early surgical intervention is strongly advised when multiple extrahepatic cysts are present and rupture is evident”, but questionably choose to discharge the patient with ABZ. It is likely that the second rupture may have been triggered by medical treatment. We agree with the authors in the choice of a sequential surgical approach prioritizing lung cysts: in multi-organ echinococcosis, due to the higher risk of complication of lung disease, thoracic surgery should be scheduled as quickly as possible and before handling abdominal disease. In some instances, i.e. parasitic cysts of the right liver with concomitant supradiaphragmatic lung cysts, same-time surgery (chest and liver) can be performed by experienced surgeons. Otherwise, surgery in two times is an acceptable option, unless in case of complicated abdominal cysts. The authors adequately chose a parenchymal-sparing technique, according to expert recommendations 12. No information is given about the use of surgical field protection (pads soaked with hypertonic saline) and, more importantly, about the management of the left lung cyst complicated by rupture: as highlighted above, pleural fistula requires surgical toilette, otherwise the patient is at high risk of secondary echinococcosis. We are interested in knowing how did the authors deal with the left lung ruptured cyst. The authors said that the patient was discharged with oral ABZ after the first surgery for lung disease, but we did not find details about the timing of coverage and if the ABZ prophylaxis was used for the subsequent abdominal surgery: according to recommendations, a 1-3 months course of ABZ after each surgical act is required. Some remarks about the Introduction:\nIncidence of CE (number of new infections/year) is difficult to calculate because the disease lacks an acute symptomatic phase after infection and therefore can go unnoticed. The surrogate epidemiological parameter is the number of new diagnosis/year, based on symptomatic cases seeking medical care. Unfortunately, this measure does not reproduce the real burden of disease, because a great majority of CE cases are asymptomatic, hence not reported. We suggest to change the reference n.1 in relation with CE epidemiology and mortality rate, since the paper by Schweiger et al. is about alveolar echinococcosis (AE). The topic is well described in the review by Wen et al. 3.\nIn the discussion chapter:\nWe would like to comment on the sentence “a single cyst can contain numerous protoscoleces”: strictly speaking, a single hydatid fertile cyst contains hundreds to thousands of protoscoleces, as shown in studies carried out in the intermediate host 4. A more accurate description of the definitive host infection should be given: canids acquire the metacestode through ingestion of parasitized viscera of the intermediate host. Diagnosis of CE is mainly based on imaging methods. Others elements (clinical picture, laboratory examination and serology) are ancillary, but not conclusive without imaging 5. Microscopic evidence of protoscoleces in cyst fluid taken with surgery or aspiration allows direct diagnosis, although not always feasible or essential. CE is not a frequent cause of eosinophilia, but when eosinophilia is detected in a patient from an endemic area, CE should be considered as eosinophilia suggests leakage or rupture 6.\nTreatment of CE is complex and indications are different in lung and liver disease. In liver CE and broadly speaking in abdominal CE, surgery is not necessarily the best option and should be based primarily on a stage-specific approach, as per WHO recommendations 1:\nCE1 and CE3a liver cysts do not need necessarily need surgery and can be managed with ABZ or with percutaneous treatments, according to their size; CE4 and CE5 liver cysts do not need any treatment if uncomplicated. They need only ultrasound follow-up to capture relapses (watch and wait); CE2 and CE3b stage are cured by surgery, as they respond poorly to medical or percutaneous treatments other than Modified Catheterization techniques; Complicated liver cysts (i.e. biliary fistula, infected cysts) require surgery.\nConversely surgery is the only treatment for lung CE with prompt partial cystectomy as first option 5. In the colleague’s paper no information about the cyst stage is available and this point is noteworthy and should be addressed because it is crucial for choosing the treatment. Apparently, an ultrasound examination of liver and splenic cysts has not been performed, hence cysts stage is lacking. In this case, if abdominal cysts were active cysts (CE1 stage), they could have potentially been treated with medical or percutaneous treatment, turning to surgery once conservative therapy had failed. We read that laparoscopic endocystectomy was performed on liver and splenic cysts. It would be useful to share details of the procedure on the spleen: was spleen removed together with the cyst? Spleen-sparing surgical options need to be considered because splenectomized patients are at significant risk of infection, with overwhelming post-splenectomy sepsis (OPSI) as the most serious complication (vulnerability to invasive infections by encapsulated bacteria: S. pneumoniae, H. influenzae, N. meningitidis). Prevention with vaccination against such pathogens is recommended 7. Finally, it would be useful to know if clinical and radiological follow-up is planned: if available, we would appreciate to evaluate imaging of lungs and abdomen after surgery.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "11333", "date": "04 Apr 2024", "name": "Amro Abdelrahman", "role": "Author Response", "response": "Dear reviewer, Thanks for feedback. It would be more practical if you rewrite your points in a bullet point writing style." } ] }, { "id": "224736", "date": "14 Feb 2024", "name": "Khawaja Bilal Waheed", "expertise": [ "Reviewer Expertise Radiology 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\nThe authors did not mention why the radiologist suggested a PAN CT. Also, while providing a caption for CT scan images, please correct the caption as it is not PAN CT but rather selected axial, and coronal images in lung and abdominal windows.\nThe authors did not provide differential diagnosis for this case, both at initial presentation or in discussion part.\nIt would be better to include a histopathological specimen picture or slide demonstrating the hydatid cyst.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "11331", "date": "04 Apr 2024", "name": "Amro Abdelrahman", "role": "Author Response", "response": "Dear reviewer. Thanks for feedback. We will add that pan ct was recommended to look for other cystic involvemen in othe organs. We will correct the figure issue. We will add differential diagnosis in case presentation Thanks again," } ] } ]	1	https://f1000research.com/articles/12-1492
https://f1000research.com/articles/12-1154/v1	14 Sep 23	{ "type": "Clinical Practice Article", "title": "Severe meibomian gland loss in polycystic ovarian syndrome patients on estrogen-progesterone therapy: A case series", "authors": [ "Japmehr Kaur Sandhu", "Swati Singh", "Sayan Basu", "Japmehr Kaur Sandhu", "Sayan Basu" ], "abstract": "Purpose: To report the ocular surface and meibomian gland changes in polycystic ovarian syndrome (PCOS) women taking hormone supplementation.\nMethods: Case series.\nResults: Three women (27 ± 11 years) already diagnosed with PCOS presented with dry eye symptoms (mean OSDI, 37.5) for a mean duration of 13 months and were taking hormonal supplements for a mean duration of 60 ± 11 months. The hormonal supplements included oral estrogen (n=3), oral progesterone (n=3), antiandrogen cyproterone (n=1) and isotretinoin (n=1). Ocular surface evaluation revealed mean NIBUT of 9.9 ± 1.6 seconds and mean TMH of 0.27 ± 0.05 mm assessed non-invasively using Oculus keratograph 5M (K5M). Meibography (K5M) showed near total loss of all meibomian glands (n=8/12 eyelids) with residual ghost glands in all four eyelids of two patients, and gland shortening alone in one eyelid. The gland morphology did not change following intense thermal pulsation treatment or cessation of hormonal therapy.\nConclusions: Near-total irreversible meibomian gland loss was seen in two young PCOS women taking hormonal supplements. Collaboration between ophthalmologists and gynecologists is advisable for early detection and better understanding of dry eye disease (DED) progression in these patients.", "keywords": [ "Meibomian gland", "Dry eye disease", "Sex Hormones", "Hormone replacement therapy" ], "content": "Introduction\n\nPolycystic ovarian syndrome (PCOS) is one of the commonest endocrine abnormality in women of reproductive age. An association between PCOS and ocular surface alterations has been proposed due to hormonal imbalance and hyperandrogenism seen in PCOS.1–6 Many components of the ocular surface are influenced by sex hormones, including the lacrimal gland, meibomian glands, and cells of the conjunctiva and cornea.7 In PCOS, increased mucus production, allergy-like symptoms and conjunctival inflammation have been observed.4 Published data has shown increased meibomian gland dysfunction (MGD) in PCOS that had no correlation with testosterone levels.1 The reported changes in meibomian glands in PCOS were average 20% loss in meibomian gland area in the lower eyelid, elevated OSDI score, and reduced tear stability.1–6 Meibomian gland epithelial cells have been shown to express sex-steroid hormone receptors (especially estrogen, androgen) that affect lipid synthesis within the glands. However, the effects of hormone replacement therapy (HRT, estrogen and progesterone) on meibomian gland function of post-menopausal women are variable, with few reporting improvements, while others reported worsening with HRT.8,9 To the best of our knowledge, the effects of HRT on the ocular surface of PCOS women have not been reported before. The authors present three such cases where severe meibomian gland loss and dry eye disease (DED) was observed in women with PCOS taking prolonged hormonal supplementation.\n\n\nCase series\n\nThree women (mean age, 27±2 years) of Indian origin diagnosed with meibomian gland dysfunction who were taking hormone supplements for polycystic ovarian dysfunction were included (Figure 1). One of the women was a homemaker and the rest two were students. There was no significant family history of autoimmune DED. The chief complaints were dryness/irritation (n=2), pain (n=1), burning sensation (n=2), floaters (n=1) and a ‘pulling sensation’ in both eyes (n=1). All had bilateral ocular involvement with DED symptoms. The mean duration of ocular symptoms was 13 months, and they were using hormone therapy for the mean duration of 60±12 months. Two were using oral estrogen and progesterone prescribed for their polycystic ovarian disease (PCOD) and one was a chronic user of isotretinoin along with oral estrogen and progesterone (Case 2; cyproterone (2 mg) and ethyl estradiol (0.035 mg)) for acne with PCOD.\n\nTable summarizes details of three PCOS patients along with tear film details.\n\nNIBUT=Non-invasive tear break-up time; TMH=Tear meniscus height; OSS= Ocular staining score; F=Female; * indicates grading system by Arita et al. 2016.\n\nMeibography showed severe gland loss in both eyes of two patients (Figure 1; Case 1 and 2) whereas moderate loss in one patient (Figure 1; Case 3). The changes were equally distributed in the upper and lower eyelids. In 8/12 eyelids, the glands had a ghost-like appearance where only the terminal ductal opening was hyperreflective in few of them. The remaining visible glands were thinned out in these two patients.\n\n\nTear film and ocular surface changes\n\nThe mean non-invasive tear break-up time (NIBUT) was 9.9 ± 1.6 seconds. None of the eyes had aqueous deficiency as mean tear meniscus height (TMH) value was 0.27 ± 0.05 mm and mean Schirmer without anesthesia was 24 ± 10 mm (Figure 1). Meibomian glands were not expressible in two patients and clear meibum in third patient (Case 3). Ocular surface did not show any congestion but had ocular staining score of 1 in all of them. One of the patients had lagophthalmos of 2 mm and inferior corneal scarring (Case 2).\n\nAll patients were put on sodium hyaluronate 0.18% eye drops. Case 3 with DED and exposure keratopathy changes was prescribed additional eye drops (containing hydroxypropyl methylcellulose 0.3%, dextran 70 0.1%, glycerin 0.2%). Case 2 underwent lipiflow therapy but showed no improvement in symptoms or meibomian gland morphology following treatment. The patients were referred to gynecologist for their respective disorders and dosing schedule of hormonal supplementation.\n\n\nDiscussion\n\nThe present paper describes the severe meibomian gland loss observed in three PCOS women taking hormonal supplementation. Interesting findings in two of the cases were near total loss of meibomian glands in upper and lower eyelids of both eyes with near normal tear break-up time (9 s). This is in contrary to the reported tear film changes seen in PCOS who are not on any hormonal therapy. Almost complete loss of meibomian glands from all eyelids has been rarely reported on meibography in young PCOS women (mean age, 27 years) whether with or without hormonal supplementation. Other than hormonal imbalance of PCOS, oral estrogen intake that downregulates the cell proliferation within the meibomian gland acini and compete with androgens to act on the glands could be responsible for severe loss. One of the patients with acne and PCOS was taking antiandrogens, thus contributing to the gland loss. However, the morphology of gland loss was similar to the women not taking any antiandrogens. Gynecologists can be sensitized to explore DED symptomatology in PCOS patients where they are planning to administer HRT and during follow-up. A detailed ocular surface examination prior to therapy initiation would be helpful. Also, meibomian gland targeted therapies might not be advisable in women taking hormonal supplementation with almost total gland loss as they do not grow back.\n\nThe effects of HRT on tear glands are reported mostly in terms of symptoms without much knowledge about the structural changes. We found significant gland loss in our series. DED observed in menopause is supposed to be secondary to the reduced androgens levels in menopause.7 However, PCOS has hyperandrogenism and theoretically it should not result in meibomian gland loss. In the current series, all patients were taking estrogen supplements. The intake of estrogens affects testosterone levels and reduces sebaceous gland growth in the body and decrease sebocyte differentiation via the premature release of lysosomal enzymes. This might be the possible mechanism of HRT action on the meibomian glands in PCOS. Dry eye symptoms are reported in PCOS women, and they have normal Schirmer values and a negative correlation between the gland dropout and plasma estradiol levels.1 In PCOS, hyperandrogenism is expected (which supports acinar proliferation), however, these patients were taking combined hormone therapy and had meibomian gland loss. In PCOS, contact lens intolerance was reported due to increased mucus production.4 In our series, we did not observe presence of mucus threads in the tear lake, which could be due to counteracting effect of HRT. It was surprising to see normal NIBUT values despite extensive loss of meibomian glands in our series. Future studies comparing goblet cell density with other tear film parameters would need to test the hypothesis of whether increased mucus production stabilizes the tear film in these patients.\n\nThe majority of studies have refuted any role of HRT in developing dry eye symptoms but the studied interventions (hormonal supplementation) were given only for a duration of one month. Uncu et al. reported that DED symptoms start after 12 months of HRT.9 Hence, it may be postulated that prolonged use of HRT may exacerbate symptoms. In our study, women were on hormone therapy for an average duration of five years. In our series, lacrimal gland function was unaffected in all patients whereas meibomian gland loss was noted in all of them. While two patients (Case 1, 2) were on combined estrogen-progesterone therapy, Case 3 was using only norethindrone acetate (synthetic progesterone) 5 mg daily and had gland shortening compared to gland loss seen in rest of the cases. Oral intake of isotretinoin can also affect sebaceous secretions and cause atrophy of sebaceous glands.10 Rather than gland dropout, isotretinoin use is frequently associated with loss in meibomian gland density and size. Case 2 who was on chronic isotretinoin demonstrated normal Schirmer and NIBUT values but showed extensive meibomian gland dropout in both eyes. The pre-HRT therapy meibography images are not available (though all were asymptomatic earlier), hence the timeline of progression of meibographic changes and their relationship with ovarian disease or therapy could not be studied. Nevertheless, observation of near total gland loss and a detailed ocular surface and dry eye work up are the contributions of this case series.\n\nMeibomian gland dropout represents end-stage acinar atrophy leading to the loss of functional glandular tissue inside the tarsal plates, that cannot be reversed with any gland targeted therapies like Lipiflow. Follow-up in one case (Case 2) demonstrated no improvement after two months of discontinuing hormone therapy. Hence, this permanent and severe dropout requires early detection and further investigation of the relationship between the dose or duration of HRT and gland changes. The use of prolonged hormonal supplementation and antiandrogens in PCOS can be associated with severe gland atrophy as noted in our case series. Hence, a careful ophthalmological check-up might be advisable for these women before and during the hormonal therapy especially when they have DED symptoms. As meibomian gland loss is irreversible, an early timely intervention might help in preventing the gland loss. Future studies can explore the correlation between the dosage/duration of hormonal therapy and progression of meibomian gland changes on meibography.\n\n\nEthics approval\n\nWaived as this was a retrospective study.\n\n\nConsent for publication\n\nWritten informed consent was obtained from all subjects for the use of their electronic data for publication purpose.\n\n\nAuthors’ contributions\n\nAll authors contributed to the conceptualization, data collection and manuscript preparation (writing and editing).", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nBaser G, Yildiz N, Calan M: Evaluation of Meibomian Gland Dysfunction in Polycystic Ovary Syndrome and Obesity. Curr. Eye Res. 2017; 42(5): 661–665. PubMed Abstract \| Publisher Full Text\n\nBonini S, Mantelli F, Moretti C, et al.: Itchy-dry eye associated with polycystic ovary syndrome. Am. J. Ophthalmol. 2007; 143: 763–771.e2. Publisher Full Text\n\nAcet Y, Sarikaya S: Tear film impairment and meibomian gland loss in patients with polycystic ovary syndrome. Int. Ophthalmol. 2023; 43(3): 795–805. PubMed Abstract \| Publisher Full Text\n\nMantelli F, Moretti C, Macchi I, et al.: Effects of Sex Hormones on Ocular Surface Epithelia: Lessons Learned From Polycystic Ovary Syndrome. J. Cell. Physiol. 2016; 231(5): 971–975. PubMed Abstract \| Publisher Full Text\n\nAsfuroğlu Y, Kan Ö, Asfuroğlu M, et al.: Association Between Dry Eye and Polycystic Ovary Syndrome: Subclinical Inflammation May Be Part of the Process. Eye Contact Lens. 2021; 47(1): 27–31. PubMed Abstract \| Publisher Full Text\n\nBalıkçı AT, Ulutaş HG, Özgen GA: Evaluation of meibomian gland morphology and anterior segment parameters by Sirius topography systems in polycystic ovary syndrome. Indian J. Ophthalmol. 2022; 70(8): 2922–2928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocha EM, Mantelli F, Nominato LF, et al.: Hormones and dry eye syndrome: an update on what we do and don’t know. Curr. Opin. Ophthalmol. 2013; 24(4): 348–355. Publisher Full Text\n\nLiu C, Liang K, Jiang Z, et al.: Sex hormone therapy’s effect on dry eye syndrome in postmenopausal women: A meta-analysis of randomized controlled trials. Medicine (Baltimore). 2018; 97(40): e12572. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUncu G, Avci R, Uncu Y, et al.: The effects of different hormone replacement therapy regimens on tear function, intraocular pressure and lens opacity. Gynecol. Endocrinol. 2006; 22(9): 501–505. PubMed Abstract \| Publisher Full Text\n\nDüzgün E, Özkur E: The effect of oral isotretinoin therapy on meibomian gland morphology and dry eye tests. J. Dermatolog. Treat. 2022; 33(2): 762–768. Publisher Full Text" }	[ { "id": "206936", "date": "13 Dec 2023", "name": "Jaime David Martinez", "expertise": [ "Reviewer Expertise Ophthalmology", "Cornea", "Ocular surface diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoy reading this paper. It does teach the need for ophthalmologists and gynecologists to raise awareness on the causes of dry eye as they mention once meibomian glands atrophy, they do not regenerate. I'd like you to recommend the following changes.\nParagraph 1 in the discussion's sentence recommends changing the wording. “Also, meibomian gland targeted therapies might not be advisable in women taking hormonal supplementation with almost total gland loss as they do not grow back.” Change the word grow back to regenerate. “Also, meibomian gland targeted therapies might not be advisable in women taking hormonal supplementation with almost total gland loss as they do not regenerate.”\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": [] }, { "id": "281736", "date": "27 Jun 2024", "name": "Swaminathan Sethu", "expertise": [ "Reviewer Expertise Immunology", "Dry Eye Disease", "Ocular diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors with this case series highlight the relevance of a multidisciplinary team and approach in the management of PCOS/PCOD to ensure quality of life for the patient. Hormonal imbalance affects ocular surface homeostasis, in addition to other organs and tissues. Hence, it would be beneficial for patients with PCOS to be simultaneously managed by both gynaecologist and an ophthalmologist. In addition to the case details, the authors have communicated a very balanced discussion.\nThe following amendments would further help in improve readership understanding\nRephrasing the following sentence would improve the clarity of the statement made. “The reported changes in meibomian glands in PCOS were average 20% loss in meibomian gland area in the lower eyelid, elevated OSDI score, and reduced tear stability”. Suggestion if appropriate: “The ocular surface changes in PCOS patients include increased OSDI score, reduced tear film stability along with a loss of meibomian glands (~20%)” It will be useful to add a citation to the statement “Meibomian gland epithelial cells have been shown to express sex-steroid hormone receptors (especially estrogen, androgen) that affect lipid synthesis within the glands”. Polycystic ovarian syndrome, polycystic ovarian dysfunction and polycystic ovarian disease has been interchangeably used in this manuscript. Polycystic ovarian syndrome and polycystic ovarian disease are different clinical entities. In the introduction the authors have mentioned it as PCOS and in the demographics & history section it is stated as PCOD. It will be useful if the authors can clarify this aspect. The abstract includes “cessation of hormonal therapy”, however, the details pertaining to this aspect is missing in the “treatment” section of the document. It is mentioned in the discussion section that only subject \"case 2\" had undergone discontinuation of hormone therapy.\n\nSimilarly, intense thermal pulsation treatment was only done in 1 subject. It would be useful to include the regimen followed and details on how long after the intense thermal pulsation treatment was the improvement assessed. It would be helpful for the readers, if any information can be shared with reference to the reason underlying and choice of subject for (a) Cessation of hormonal therapy of 1 subject, and (b) intense thermal pulsation treatment for another subject. If possible, include clinical information (such as those listed in table insert in Figure 1) following ophthalmic care. It can be useful for the readership.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1154
https://f1000research.com/articles/12-493/v1	15 May 23	{ "type": "Study Protocol", "title": "Comparative evaluation and co-relation in variation of curve of Spee and curve of Wilson in Class II div. 1, Class II div. 2, and Class III as against Class I malocclusion in central India population- an in vitro study", "authors": [ "Ruchika Pandey", "Ranjit Kamble", "Ranjit Kamble" ], "abstract": "Introduction- Getting acceptable cosmetic results in the soft tissues of the face serves as the foundation for orthodontic treatment planning. Also, in order to achieve healthy static and dynamic occlusal interactions, the teeth must be positioned within the basal bone at the correct position, angle, and inclination. To avoid periodontal issues, provide stability, and achieve a functional occlusion, it is essential to ascertain the individual's dental arch form before starting of treatment and thus to utilise the mechanics that follow throughout the treatment.\nObjectives- To evaluate and compare variation in Curve of Spee and Curve of Wilson in Class II Div.-1, Class II-Div-2 and Class-III as against Class I malocclusion in central India population.\nMethodology- Irreversible hydrocolloid impression will be taken with perforated metal stock trays and stone cast will be poured. This will be scanned using CAD CAM machine and curve of Spee and Wilson will be measured using reverse engineering.\n\nExpected Result- It will assist us in treatment planning for preventing periodontal issues, assuring stability, and achieving functional occlusion by evaluating and comparing the Spee and Wilson curves in Class II Divison-1, Class II Divison-2, and Class-III malocclusion with Class-I malocclusion.\nConclusion- Every single patient receiving orthodontic treatment has the COS, which is crucial to achieving a stable occlusion. Almost every patient who receives orthodontic treatment eventually experiences the Spee Curve. Since there aren't many studies examining the relationship between the Curves of Spee and Wilson, their impact on dentoskeletal morphology, and their role in occlusal stability.", "keywords": [ "Curve of Spee", "Curve of Wilson", "Occlusion", "Functional occlusion" ], "content": "Introduction\n\nPlanning for orthodontic treatment begins with achieving acceptable cosmetic outcomes in the soft tissues of the face. In order to achieve healthy static and dynamic occlusal interactions, it is also crucial to place the teeth in the basal bone with the correct angulation, inclination, and location. In order to prevent periodontal disease, provide stability, and achieve a functional occlusion, it is critical to identify the patient’s dental arch form before commencing treatment so that mechanics can be used to conform arch form throughout the therapy. Periodontal problems in teeth are caused by teeth moved orthodontically that go beyond the basal border and compromise the stability after treatment.1 The occlusal plane should be flattened and the Wilson Curve should be levelled as the end results of orthodontic therapy.2\n\nThe human dentition exhibits the natural phenomenon known as Curve of Spee (COS). For a successful masticatory system, the anteroposterior curve of occlusion is necessary.2 In 1890, F. Graf von Spee (1855-1937), German anatomist, described the Curve of Spee. He discovered that the occlusion line in skulls with worn away teeth was a line on a cylinder perpendicular to the mandibular incisors’ incisal borders, surface of occlusal of 2nd molar, and the anterior border of the condyle.3 The COS, which is essential to attaining a stable occlusion, is present in every single patient undergoing orthodontic treatment. In orthodontics, the arc that is perpendicular to the incisal margins is currently referred to as the “Curve of Spee”.4\n\nRecent studies have shown that the mediolateral curve plays a biomechanical role in mastication by improving the efficacy of forces of occlusion during mastication and the crush/shear ratio between the posteriors. Dental malocclusions with significant overbites typically have an inflated Curve of Spee.3 As a result, there will be an inappropriate functional occlusion since the muscle imbalance will be altered.\n\nWilson was the very first to distinguish the lower grinding teeth’s lingual inclination and the higher grinding teeth’s buccal inclination. The Curve of Wilson is the name given to this occlusal curve in the coronal plane. The Wilson Curve allows for lateral mandibular excursions without posterior obstructions.2\n\nFor adequate function, the amount of buccolingual tooth inclination must be determined and quantified in order to support treatment objectives.2 The palatal and buccal cusps of the posteriors are in functional contact as a result of this curvature, which appears to necessitate a concave mandibular arch as well as a concave and convex maxillary arch.5 It would be logical to expect that the incline of bone would be oriented in this direction for the best masticatory stress given the alignment of the anatomical parts outlined by Dawson. Okeson outlined the purpose of the Curve of Wilson, which prevents balancing interferences and ensures the most efficient utilisation of cuspal contacts.2\n\nAndrews came up with 6 keys of normal occlusion out of which 3rd key is tooth inclination, which is the labiolingual as well as buccolingual inclination of the crown. He saw a lingual tilt in the posterior crowns of the mandible and maxilla.2 One of the elements included in the six keys to occlusion is the Curve of Spee. According to Andrews, COS in people with proper occlusion varies from flat to moderate.1\n\nThe COS and COW in the lower arches of people living in central India will be determined in this study. In this study, reverse engineering is used to determine the Wilson curve and the Spee curve, which is useful for understanding its comprehensiveness as well as the impact it has on stable occlusion. The measurement and significance of compensatory curves have also been discussed in various studies.\n\nIn literatures prior to our study, some of the compensatory curves were shown to be correlated with dentoskeletal malocclusions and normal occlusion. The link between the Curves of Spee and Wilson and their effect on morphology of dentoskeletal, their potential to maintain stability of occlusion have all been the subject of a few studies. This investigation investigates the connection between compensatory curves and how it might aid in the development of treatment plans for specific malocclusions.\n\n\n\n• To evaluate Curve of Spee and Curve of Wilson in Class II Div.-1, Class II Div.-2 and Class-III malocclusion in central India population.\n\n• To evaluate Curve of Spee and Curve of Wilson in Class-I malocclusion in central India population.\n\n• To compare variation in Curve of Spee and Curve of Wilson in Class II Div.-1, Class II Div.-2 malocclusion and Class-III as against Class I malocclusion in central India population.\n\nTrial design: In-vitro study\n\n\nProtocol\n\nFollowing study will be done at the Department of Orthodontics and Dentofacial Orthopedics, Sharad Pawar Dental College, Sawangi (M), Wardha, in cooperation with the Department of Prosthodontics.\n\nInclusion criteria:\n\n• Patients of age group between 14-30 years.\n\n• Complete permanent dentition except third molars considering it is the tooth that is most frequently lost owing to extraction or congenital tooth loss.\n\nExclusion criteria:\n\n• Patient with previous orthodontic treatment.\n\n• Patient with ongoing orthodontic treatment.\n\n• Patient with missing or impacted teeth.\n\n• Patient with systemic disease.\n\n• Patient with previous record of trauma or surgery.\n\n• Severe caries.\n\n• Severe occlusal wear.\n\n\n\n• The study will be conducted on patients coming to the Department of Orthodontics and Dentofacial Orthopedics and diagnosed with Class I, Class ІІ div-1 and Class II div-2 & Class III malocclusion will be selected for the study.\n\n• Written informed consent will be obtained from all the participants.\n\n• Irreversible hydrocolloid impression will be taken with perforated metal stock trays and stone cast will be poured. This will be scanned using CAD CAM machine and curve of Spee and Wilson will be measured using reverse engineering. Reverse engineering is the practice of disassembling an object in order to discover how it functions. It is generally done to analyze and learn how something functions, although it is frequently used to copy or improve the end result.\n\n• The curve of Spee will be measured by creating a tangent line connecting distobuccal cusp of mandibular 2nd molars and highest tip of the mandibular incisors in a sagittal section. Measurements will be taken from that tangent line to the deepest point on the premolar region (Figure 1).6\n\n• Frontal sections will be cut in the area of centre of 1st molar. We shall follow the axis of molar, which follows a line that connects molars’ furcation and occlusal groove, to measure the Wilson curve’s angle. The greatest protuberance of the soft tissues present on buccal alveolar crest, the WALA points on the right and left bones, will be used to form a reference line. We can use it to calculate the angle between the left and right molars. The total angulation will be the sum of θ1 and θ2 (Figure 2).5\n\nTo obtain a functional occlusion, it is essential to identify the patient’s dental arch form before the procedure and to apply mechanics that conform with the arch form throughout the therapy.\n\nFormula for sample size when comparing two means\n\nWhere;\n\nZα represents the degree of significance at 5% i.e., 95%\n\nConfidence interval = 1.96\n\nZβ is the test’s power = 80% = 0.84\n\nδ1 = SD of COS depth in Class I = 0.717\n\nδ2 = SD of COS depth in Class II = 0.732\n\n∆=Distiction betweentwomeans=2.472–1.954=0.518\n\nn = 30 patients needed in each group\n\nTotal sample size is 120\n\nStudy Reference: IIknur Veli et al.7\n\nAll the results will be calculated using R version 4.2.3 (Shortstop Beagle). Data for outcome variables will be tested for normality using Kalmogorov-Smirlov. Comparative analysis over the outcome of functional occlusion in different malocclusion will be evaluated and measurement of depth of curve of Spee and Wilson in millimeters respectively. ANOVA will be used to find significant difference in mean in comparison of 4 groups. Tukey test will be used for comparative evaluation of measurement in between 2 groups pairwise. P-value ≤ .05 will be considered significant 5% level of significance and 95% confidence of interval.\n\nThis protocol will assist us in evaluating and comparing the Spee and Wilson curves in Class II Div-1, Class II Div-2, & Class III malocclusions as against Class-I malocclusions, & this will help us design our treatments in order to avoid periodontal issues, ensure stability, and achieve effective occlusion.\n\nNot started yet.\n\n\nDiscussion\n\nDentists may use Spee curve analysis to predict how the occlusion will develop in the sagittal plane. The inclination of the masseter muscle was positively correlated with the Spee curve. The placement of the mandibular posterior teeth with this forward tilt increases the efficacy of the chewing muscles. In Wilson curve, the posterior teeth’s inclination is caused by two factors. The first has to do with loading resistance, and the second, with masticatory function.8\n\nSeveral of the compensatory curves have been linked to normal occlusion and dentoskeletal malocclusions in literature before our study. A few studies have looked at the relationship between the Curves of Spee and Wilson, their impact on dentition, and their capacity to preserve stability of occlusion. This study explores the relationship between compensatory curves and how it might help in formulating treatment regimens for particular malocclusions.\n\nEthical approval received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha\n\nIEC reference number - DMIHER (DU)/IEC/2023/569", "appendix": "Data availability\n\nNo source data are associated with this article.\n\nZenodo. Comparative evaluation and co-relation in variation of curve of Spee and curve of Wilson in class II div-1, class II div-2, and class III as against class-I malocclusion in central India population- an in vitro study. DOI: 10.5281/zenodo.7816557.\n\nFile Name: SPIRIT_checklist RUCHIKA.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nI would like to thank my institute and my colleagues.\n\n\nReferences\n\nDindaroğlu F, Duran GS, Tekeli A, et al.: Evaluation of the Relationship between Curve of Spee, WALA-FA Distance and Curve of Wilson in Normal Occlusion. Turk. J. Orthod. 2016 Dec; 29(4): 91–97. PubMed Abstract \| Publisher Full Text\n\nYang B, Chung CH: Buccolingual inclination of molars in untreated children and adults: A cone beam computed tomography study. Angle Orthod. 2019 Jan; 89(1): 87–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar KPS, Tamizharasi S: Significance of curve of Spee: An orthodontic review. J. Pharm. Bioallied Sci. 2012 Aug; 4(Suppl 2): S323–S328. PubMed Abstract \| Publisher Full Text\n\nDhiman S: Curve of Spee - from orthodontic perspective. Indian J. Dent. 2015; 6(4): 199–202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarrera JM, Llamas JM, Espinar E, et al.: Wilson maxillary curve analyzed by CBCT. A study on normocclusion and malocclusion individuals. Med. Oral Patol. Oral Cir. Bucal. 2013 May 1; 18(3): e547–e552. Publisher Full Text\n\nBabu KS, Kumar AN, Kommi PB, et al.: Evaluating the correlation between anteroposterior and mediolateral compensatory curves and their influence on dentoskeletal morphology-an in vitro CBCT study. J. Clin. Diagn. Res. 2017; 11(8): ZC49–ZC52. PubMed Abstract \| Publisher Full Text\n\nVeli I, Ozturk MA, Uysal T: Curve of Spee and its relationship to vertical eruption of teeth among different malocclusion groups. Am. J. Orthod. Dentofacial. Orthop. 2015 Mar; 147(3): 305–312. PubMed Abstract \| Publisher Full Text\n\nAl-Qawasmi R, Coe C: Genetic influence on the curves of occlusion in children seeking orthodontic treatment. Int. Orthod. 2021 Mar; 19(1): 82–87. PubMed Abstract \| Publisher Full Text" }	[ { "id": "255515", "date": "01 Apr 2024", "name": "Luz Andrea Velandia Palacio", "expertise": [ "Reviewer Expertise Orthodontics and dentofacial orthopedics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 underscores the significance of the Curve of Spee (COS) and Curve of Wilson (COW) in relation to occlusion and mastication, as well as their biomechanical functions. It further explores the correlation between these curves and dental malocclusions, their influence on treatment planning, and their connection to dentoskeletal morphology and occlusal stability. The aim of the study is to investigate the connection between compensatory curves and how it might aid in the development of treatment plans for specific malocclusions . I would like to point out the following in my review of this manuscript:\n\nThe objectives are “to evaluate the curve of Spee and the curve of Wilson in Class II Div.-1, Class II Div.-2, and Class-III malocclusion in the central Indian population. To evaluate the curve of Spee and the curve of Wilson in Class-I malocclusion in the central Indian population.” corresponds to the steps followed in the procedure of the study, not the aims of the study. Perhaps it would be better to state that the study will identify the relationship between the Spee curve and the Wilson curve for each malocclusion type. It is not clear if the measurements will be done by one or more observers; if so, it should be specified that an interrater reliability test will be performed and information regarding the observers experience in the field. Explain the lack of a direct 3D scan of the patients, and keep in mind the differences in accuracy between direct scanning and cast model scanning. In the Introduction section, where there is the following statement:” The occlusal plane should be flattened and the Wilson curve should be levelled as the end results of orthodontic herapy,\" reference number 2 is used incorrectly. The author who made that original statement corresponds to [1] Please correct.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11430", "date": "29 Apr 2024", "name": "Ruchika Pandey", "role": "Author Response", "response": "Thankyou sir for your thorough analysis of my article. I have made the following changes based on your review. 1. The objectives are “to evaluate the curve of Spee and the curve of Wilson in Class II Div.-1, Class II Div.-2, and Class-III malocclusion in the central Indian population. To evaluate the curve of Spee and the curve of Wilson in Class-I malocclusion in the central Indian population.” corresponds to the steps followed in the procedure of the study, not the aims of the study. Perhaps it would be better to state that the study will identify the relationship between the Spee curve and the Wilson curve for each malocclusion type. Response- I have done the necessary changes. 2. It is not clear if the measurements will be done by one or more observers; if so, it should be specified that an interrater reliability test will be performed and information regarding the observers experience in the field. Response -I have specified about interrater reliability test. 3.Explain the lack of a direct 3D scan of the patients, and keep in mind the differences in accuracy between direct scanning and cast model scanning. Response - Regarding the lack of a direct 3-D scan of the patient, scanning intraorally will provide a more accurate result, however due to the unavailability of an intraoral scanner in our location, we will have to scan the cast instead. We will do our best to eliminate distortion while taking and pouring the impression cast. 4.In the Introduction section, where there is the following statement:” The occlusal plane should be flattened and the Wilson curve should be levelled as the end results of orthodontic herapy,\" reference number 2 is used incorrectly. The author who made that original statement corresponds to [1] Please correct. Response - I have done the correction." } ] }, { "id": "236704", "date": "27 Jun 2024", "name": "Vabitha Shetty", "expertise": [ "Reviewer Expertise Sleep dentistry", "preventive dentistry", "cephalometric 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\nAn interesting study, especially useful in orthodontic rehabilitation of patients.Background, rationale and objectives are well written. The study protocol seems well thought about and structured. Sample size seems appropriate and satisfactory. Could the authors state the reasons they have chosen the CAD CAM machine for analysis of cast ? What are the advantages?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11914", "date": "27 Jun 2024", "name": "Ruchika Pandey", "role": "Author Response", "response": "Thankyou Ma'am for thorough analysis of my article. Ma'am I am well aware that direct 3-D scanning intraorally produces a more precise result; however, due to the lack of an intraoral scanner in our location, we will have to scan the cast instead. Also, ma'am, we are employing the best CAD-CAM equipment, Dentsply Sirona. It has the essential feature of allowing you to work on machine-readable measurements and geometry, which is required for our investigation. Furthermore, it is highly precise, simple to copy, change, and annotate, and can be sent directly to milling for automated production. I can also send the STL file so that competent technical engineers can review the results and measurements. Thankyou" } ] } ]	1	https://f1000research.com/articles/12-493
https://f1000research.com/articles/12-945/v1	07 Aug 23	{ "type": "Research Article", "title": "Scoutknife: A naïve, whole genome informed phylogenetic robusticity metric", "authors": [ "James Fleming", "Pia Merete Eriksen", "Torsten Hugo Struck", "Pia Merete Eriksen", "Torsten Hugo Struck" ], "abstract": "Background: The phylogenetic bootstrap, first proposed by Felsenstein in 1985, is a critically important statistical method in assessing the robusticity of phylogenetic datasets. Core to its concept was the use of pseudo sampling - assessing the data by generating new replicates derived from the initial dataset that was used to generate the phylogeny. In this way, phylogenetic support metrics could overcome the lack of perfect, infinite data. With infinite data, however, it is possible to sample smaller replicates directly from the data to obtain both the phylogeny and its statistical robusticity in the same analysis. Due to the growth of whole genome sequencing, the depth and breadth of our datasets have greatly expanded and are set to only expand further. With genome-scale datasets comprising thousands of genes, we can now obtain a proxy for infinite data. Accordingly, we can potentially abandon the notion of pseudo sampling and instead randomly sample small subsets of genes from the thousands of genes in our analyses. Methods: We introduce Scoutknife, a jackknife-style subsampling implementation that generates 100 datasets by randomly sampling a small number of genes from an initial large-gene dataset to jointly establish both a phylogenetic hypothesis and assess its robusticity. We assess its effectiveness by using 18 previously published datasets and 100 simulation studies. Results: We show that Scoutknife is conservative and informative as to conflicts and incongruence across the whole genome, without the need for subsampling based on traditional model selection criteria. Conclusions: Scoutknife reliably achieves comparable results to selecting the best genes on both real and simulation datasets, while being resistant to the potential biases caused by selecting for model fit. As the amount of genome data grows, it becomes an even more exciting option to assess the robusticity of phylogenetic hypotheses.", "keywords": [ "phylogenetics", "bootstrapping", "software" ], "content": "Introduction\n\nThe genomics revolution completely altered our understanding of phylogeny - the study of the relationships between organismal groups. By combining molecular and morphological data, our picture of the evolution of life has become clearer than ever before.1,2 We are now in the process of entering the next phase of the genomics revolution, however, where beyond single or multi-gene datasets, researchers are now able to accurately sequence whole genomes from multiple species with relative ease.3–5 This new era of “big data”, properly leveraged, promises to revolutionise our understanding of phylogenetics in the same way our prior understanding was revolutionised by the discovery of genomics itself. However, appropriately handling this new data is key to unlocking its potential. First, the robustness or statistical significance of these new results must be appropriately assessed. Second, assurances must be provided that the phylogenies reflect the actual biological processes and are not being misled by reconstructive biases.1,6,7\n\nThe robustness and reliability of a phylogenetic topology and its branches can be quantified in a number of ways, such as through Bayesian posterior probabilities8 or the Likelihood Ratio Test family of support values.9,10 One of the most common, however, is the bootstrap support value.11 A measure of statistical robustness, the bootstrap was first applied to phylogenetics by Felsenstein in 1985. In its implementation, the phylogeny is reconstructed from the limited source dataset and the bootstrap creates multiple pseudo replicates of the source datasets – effectively multiplying the signal of some sites in the datasets and removing the signal of others (Figure 1A). A variation of this approach is the generation of pseudo samples by jackknifing – resampling only a fraction of the sites (e.g., 60% or 80%) from the source dataset.12 This measures robustness, assessing how many of the sites in the source dataset support the final phylogeny – or more specifically its branches – and thereby whether there is a broader consensus for the proposed most likely topology – or for a particular branch – amongst the source dataset’s component sites.13 This method is particularly useful where data is limited, such as in single or limited gene datasets, where pseudo replicates can greatly proportionally increase the effective size, and thereby statistical power, of the analysis.11 It was originally implemented as a surrogate for a robust statistical sampling procedure from theoretically unlimited data.14 In the case of unlimited data, one could generate random samples from these data, then generate the tree of each sample and determine the overall phylogeny by including measurements of the robustness across all generated trees (Figure 1B).\n\nNote that Scoutknife bears more similarity to unlimited data sampling than a traditional bootstrap. Scoutknife may not take the same gene twice within the same sample but may take the same gene multiple times between samples – see Scoutknife replicate #1 and #2, which both sample gene #97. The structure of this figure is based upon Hillis et al. (1996), Chapter 11, page 508, Figure 33.18\n\nIn the modern era, genome-scale data is being generated for a rapidly increasing number of species across the tree of life.4,15–17 When data is plentiful, the reliance on pseudo samples becomes less necessary. With thousands of genes and millions to billions of base pairs on the horizon for phylogenetic analyses, one can safely assume that the theoretical assumption of unlimited data is not violated. Accordingly, the data can be repeatedly sampled directly, trees reconstructed and the phylogeny and statistical support determined (Figure 1C) as outlined directly from the aforementioned unlimited data.\n\nAt the same time, however, the reconstruction of the species history can be challenging due to either methodological incongruence (i.e., not all genes contain information about the species history that we can correctly decipher) or biological incongruence (i.e., not all genes follow the species history).1,6,7 Making use of large amounts of genome data comes with both an important caveat and an important boon: while methodologically incongruent genes can be removed by a number of tools to identify branch length heterogeneity, compositional heterogeneity and site saturation, genes that are excluded due to biological incongruence may contain real biological information that alters our understanding of species relationships.1\n\nCurrently, phylogenetics has adopted a conservative stance,1,18 selecting genes that fit well within our models of evolution19,20 – this has the benefit of evading artifactual topologies, but may well be presenting us with hypotheses of evolution that are preselected according to our own biases, or incorrectly causing us to adopt great confidence in hypotheses that are not as well supported by the data as it first appears.21 In this respect, an approach that is conservative towards the models may not be conservative towards confidence in our phylogenetic hypotheses. Scoutknife presents an alternative.11,22 By randomly sampling data across the genome, the key hurdle for this methodology is assessing whether methodological incongruence significantly negatively influences the final hypothesis, or whether the false signal supplied by these model violations is outweighed by the addition of the real biological signal supplied by the sheer density of big data. If the latter is true, Scoutknife may represent a better way forward for generating phylogenetic topologies – one that is robust to methodological incongruence whilst expressing the biological incongruence that is present in the data.\n\nHere we present Scoutknife, a new method for assessing topological support. In contrast to the traditional bootstrap approach, Scoutknife discards the creation of artificial pseudo replicates to instead use large multi-gene inputs to create true replicate samples from the larger pool of genes. Scoutknife is a naive and unbiased way to measure support with genome-scale data. It does this by generating a sample number of datasets, each consisting of a user-specified number of randomly selected genes and forming a consensus tree from the results (Figure 1). Alternatively, Scoutknife support values can be attached to nodes of a maximum likelihood tree, similar to conventional bootstrap support. Across 18 real and 100 simulation datasets, Scoutknife consensus trees produce comparable topological results to selecting the best genes within the dataset using GeneSortR,19 and is robust to poor data occupancy. In addition, Scoutknife proves to be more granular in its assessment of topological reliability than traditional bootstrap values, allowing researchers to be more cautious and informed about their topological hypotheses than ever before.\n\nScoutknife takes a “brute force” approach to assessing phylogenetic robusticity, simply asking the question - “how robust is the most likely tree to topological signal across the entire dataset?”. Rather than generating pseudo samples by randomly sampling sites, as in a traditional bootstrap,11 Scoutknife generates real data samples by randomly sampling genes to create randomly assembled concatenated multi-gene datasets (Figure 1C). In theory, though some of these genes contain low signal and others contain signal not consistent with the species phylogeny – either by methodological or biological incongruence1,7 – the majority should contain at least some signal of the overall species tree, thereby allowing us to more robustly quantify not only the degree of support for a given taxonomic topology, but also the degree of discordance within the constituent genomes themselves. This naïve method may further allow us to resolve new phylogenetic hypotheses that have previously been neglected due to a focus on data selection.\n\nFirst, the multi-gene dataset is divided into individual gene alignments. These alignments are then randomly selected to form 100 multi-gene partitioned datasets of a size equal to that selected by the user. The same gene cannot be selected twice by the same dataset (Figure 1C) – a key difference from a traditional bootstrap11 – but may appear multiple times across different datasets. Within our real dataset analyses, this sampling comprised 100 100-gene datasets selected from multi-gene datasets ranging from 1049 to 5105 genes (Table 1). Our simulated datasets comprised 100 replicates of a 1049 gene dataset, from which 100 100-gene datasets were then sampled.\n\n\nMaterials & Methods\n\nFor both real and simulation analyses (for details see below), 100 genes were randomly selected 100 times from the source datasets, generating 100 100-gene concatenated sample datasets using the Scoutknife Package (https://github.com/JFFleming/Scoutknife). The Scoutknife script package requires catsequences23 to be installed as a prerequisite, available at (https://github.com/ChrisCreevey/catsequences).\n\nPhylogenies for each Scoutknife dataset were constructed under IQ-Tree v1.6.1224 using ModelFinder,25 with a separate model applied to each gene and no partition merging. As a data density-based technique, Scoutknife might be expected to perform better in high data density scenarios where partitions can be comfortably merged. As such, this was intended to limit the efficacy of Scoutknife further and test its performance under a scenario with more highly variable best fit models than might be expected under normal conditions, whilst conserving computational effort considering the large number of test datasets and simulations. To further facilitate parallelization of the analyses, the phylogenetic analyses of the datasets were submitted using Scoutknifette (https://github.com/Togtja/scoutknifette). Scoutknifette is a custom high performance computing (HPC) webhook for the group messaging service Discord that can be easily modified for any HPC tasks that require multiple submission batches and queue tracking.\n\nThe trees produced by each Scoutknife sample dataset were then concatenated into a single treelist file (see Underlying Data in our Data Availability Statement), and a consensus tree was constructed using bpcomp, available in Phylobayes,26 by using a burnin of 0 and a sampling rate of 1, sampling each tree in the treelist. Trees were constructed as both 70% strict consensus and 50% majority consensus trees, and the results were compared. In two cases (Araneae and Lepidoptera), 30% plurality consensus trees were constructed using the same method, to further explore the data, as explained in the results and discussion section. In a single case (Actinopterygii), the low occupancy of two species in particular (Muraenesox cinerus with 1 gene and Scomber scombrus with 15 genes across the entire dataset of 1105) meant that many of the Scoutknife samples did not contain representatives from these taxa. To address this, we used sumtrees.py v 4.5.2, part of the DendroPy package,27 as it is capable of building consensus trees from tree lists containing a variable number of taxa.\n\nThe Quartet Similarity, Quartet Divergence, Node Conflict, Node Agreement, Strict Joint Assertions, Semi-Strict Joint Assertions, Symmetric Difference, Marczewski-Steinhaus, Steel-Penny and Overall Similarity were measured with reference to the previously published topology of the 250 most informative genes of that dataset, as selected by GeneSortR.19 In the case of the simulated datasets, the topology of the 250 most informative genes of the original source dataset, Milla et al., (2020), as selected by GeneSortR,19,28 was used. These similarity metrics were calculated using the ‘Quartet’ Library available in R.29\n\nTo assess the efficacy of Scoutknife, we examined 18 real data datasets,28,30–46 those used in a similar benchmarking study by GeneSortR.19 These datasets range from 1049 to 5105 genes and from 30 to 332 taxa in size, comprising studies of animals, plants and fungi (Table 1). In contrast to the prior study, genes with less than 50% occupancy were not removed: Scoutknife should show decreased performance at low occupancy levels, as it relies on data density, and so this should give a clearer picture of how the methodology performs across a variety of real datasets. The resultant tree topologies were then compared to the topology recovered by analysing the most informative 250 genes, as determined by GeneSortR,19 to assess whether the same topological hypothesis was resolved by the Scoutknife Consensus Tree.\n\nThe first column names the taxa that form the ingroup of the phylogeny. The second names the original publication (although all source datasets are the same as those evaluated by Koch et al. (2021).19 The third and fourth columns detail the number of taxa and the number of genes in the original alignment respectively.\n\nTo further assess the efficacy of Scoutknife, we generated 100 simulation datasets using the Alignment Mimic function of AliSim, as implemented in IQTree v2.2.0.47,48 For this, 100 simulations were independently created for each gene in the Milla et al., (2020)28 Heliozelidae dataset, as it represented a small-sized dataset of those within our real data study, at 1049 genes, and as such should have presented a challenge for Scoutknife. Furthermore, AliSim’s alignment mimic47 allows us to generate alignment datasets that mimic real genes, complete with low occupancy and reasonable variations in alignment length. Alisim was implemented with the following command:\n\nFor each set of 1049 simulated genes, 100 100-gene Scoutknife datasets were constructed, and then analysed using IQ Tree as with the real datasets. The Quartet Similarity, Quartet Divergence, Node Conflict, Node Agreement, Strict Joint Assertions, Semi-Strict Joint Assertions, Symmetric Difference, Marczewski-Steinhaus, Steel-Penny and Overall Similarity were then measured with reference to the previously published topology generated by analysing the 250 most informative genes of the Milla et al. (2020) dataset as selected by GeneSortR.19 As each gene was simulated independently, it should in theory retain the topology of that initial single gene dataset, thereby replicating the discordance present in the original dataset. Furthermore, by directly comparing our random samples of simulated datasets to the most informative genes of the source dataset, this should disadvantage Scoutknife, as some of the simulated data may support a separate alternative topology to either the single gene or the real informative gene topology.\n\nFor each dataset, we calculated a variety of quartet-based similarity metrics: the Quartet Divergence,49 the proportion of nodes that did not conflict between trees, the proportion of nodes that explicitly agreed between trees, the proportion of strict and semi-strict joint assertions,50 the symmetric difference between trees51 and the Steel-Penny52 and Marczewski-Steinhaus similarity metrics.51 Concordance with the initial study’s topology was first measured by assessing the proportion of nodes that explicitly agreed between topologies and then the proportion of nodes that did not conflict with the recovered topology. This could then be further scrutinized using the Quartet Divergence and then the Marczewski-Steinhaus (MS) measurement, which compares the distinctly resolved quartets in common between both trees. The remaining quartet measurements are present in our Underlying Data, available at DataDryad. Robinson-Foulds (RF) distances were not used due to Scoutknife’s propensity to recover nodes with conservatively low amounts of support. Polytomies are known to bias RF distances as they rely on a completely resolved tree, and this would be incompatible with the Scoutknife approach, which explicitly favours polytomies as representations of incongruent signal in the genome.53\n\n\nResults & Discussion\n\nAcross our 18 real test datasets, on a majority consensus tree, Scoutknife only struggles to recover the topology initially recovered by the original study in two cases (indicated by an explicit agreement of nodes below 90%, Quartet Divergence greater than 5% or a Marczewski-Steinhaus below 0.9) (Figure 2). In the Araneae, Scoutknife achieved an “explicit agreement” value of 81.10%, Marczewski-Steinhaus of 0.80, and quartet divergence of 9.87%, which prompted us to further examine the dataset. The average occupancy of the dataset, when including genes with below 50% occupancy, is 46%. Furthermore, only 97 of the 2366 genes in the dataset had an occupancy greater than 80% (Figure 3). In this case, it appears that Scoutknife struggles with lower resolution data, and that large amounts of missing data may be a genuine challenge to the efficacy of the method. However, when assessed using the more liberal criteria of measuring the proportion of nodes that do not conflict with the published tree (which is a measure that accounts for the uncertainty expressed by polytomies), 99.17% of recovered nodes were found to not be in explicit conflict (Figure 2). This suggests that 18% of this discordance is caused by a conservative assessment of support in the data considering its low occupancy, not by disagreement in inference.\n\nThe two datasets discussed further in the text, Araneae and Lepidoptera, are highlighted in light blue (for non-conflict) and red (for explicit agreement) respectively.\n\nA large proportion of low occupancy genes may cause issues for Scoutknife resolution.\n\nThe second dataset that appeared to struggle under the Scoutknife approach was the Lepidoptera dataset. Here, only 81.66% of nodes were found to explicitly agree with the published topology, and it produced an MS value of 0.82 and quartet divergence of 9.19%. As in the Araneae, we find that 99.95% of the nodes did not conflict with the published topology. However, the reasons for this discordance within the Lepidoptera is less clear. This dataset had the sixth highest occupancy of the real datasets (88.8%), many of which produced more well-resolved Scoutknife consensus trees. Furthermore, GeneSortR measured the “Usefulness” of the dataset as the third highest of the selected study sets (0.33, on a range from 0.14-0.48).19 Changing the minimum consensus value to produce a Scoutknife tree from a majority consensus tree to a 30% plurality support tree increases the Marczewski-Steinhaus value to 0.93, decreases quartet divergence to 3.63% and increases the number of nodes found to explicitly agree with the published topology to 92.83% (Underlying Data). This suggests that the discordance within the Lepidoptera dataset may be a true biological property of the history of the group, and that the difference between the Scoutknife result and prior published results may be indicative of gene selection and analysis methods strongly favouring one of a series of genuine alternative hypotheses that Scoutknife prefers to represent as a polytomy. This assertion is particularly supported when contrasted against the Araneae dataset – there, changing the minimum consensus value to produce a 30% plurality support tree increases the Marczewski-Steinhaus value from 0.80 to only 0.87 (0.07 increase Araneae vs. 0.11 in Lepidoptera), decreases quarter divergence from 9.87% to 6.77% (3.1% decrease vs 5.56% in Lepidoptera), and increases the number of nodes that “explicitly agree” from 81.10% to 87.59% (6.49% increase vs. 11.17% in Lepidoptera), a much smaller overall change in comparison.\n\nIn the opposite direction, on a stricter 70% consensus tree, Scoutknife achieves an average of 99.85% nodes not conflicting with the tress produced by GeneSortR, ranging from 100% to 99.16%. At this higher value, however, explicit agreement varies between 56.91% (in the Araneae) and 99.90%, with an average of 94.35% (or 96.56% if the Araneae are excluded). This is due to the innate conservatism of Scoutknife – as the consensus guideline is increased, it is more likely to favour collapsing more nodes into polytomies – the average decrease in Explicit Agreement with the GeneSortR tree between the majority consensus trees and the 0.7 consensus tree is 2.91%, with values ranging from 0% (Echinoidea) to 24.18% (Araneae).\n\nWithin our simulation datasets, Scoutknife consistently recovers topologies that are consistent with the GeneSortR tree – the 70% strict consensus simulation trees recover no conflicting nodes with the GeneSortR topology. However, across the 100 simulation consensus trees, not all explicitly agree with the nodes resolved by GeneSortR (Figure 4). At 70% strict consensus, explicit agreement varied between 97.81% and 88.64% with an average of 92.85%. This represents the greater conservatism of Scoutknife as a method – across all analyses, it prefers to resolve as polytomies, rather than bifurcations, representing the discordance across the genes in the dataset. This is further confirmed by the Marczewski-Steinhaus similarity index, which is consistent with the explicit agreement values (varying from 0.89 to 0.98 with an average of 0.93), suggesting that the only difference between the Scoutknife and GeneSortR topologies is in the existence of polytomies.\n\nNote the long tail on the Majority Marczewkski-Steinhaus violin, representing Simulation 20.\n\nExamining the simple majority consensus trees, requiring a consensus of only 50% of resolved gene trees to resolve the node and not 70%, two bifurcating topologies were produced that conflicted with the GeneSortR topology (Simulation 20 and Simulation 98), reducing the average nodal “Do Not Conflict” value from 100% to 99.92%. While the Simulation 98 topology was very similar to the GeneSortR topology (Quartet Divergence 0.010), Simulation 20 showed significant divergence from the GeneSortR topology (Quarter Divergence 0.082).\n\nThe discordance in Simulation 20 is caused by a single node distinguishing the Pseliastis group, Hoplophanes group and the Heliozela/Antispila/Antispilina/Holocacista/Coptodisca group. The Scoutknife analysis of Simulation 20 recovers this node with a support of 0.51 for (Pseliastis+Hoplophanes), the topology that is not favoured by GeneSortR or the remaining 99 Scoutknife simulations. GeneSortR recovered the alternative topology (Pseliastis+ Heliozela/Antispila/Antispilina/Holocacista/Coptodisca) with a boostrap support of 83, the second least supported node in the entire Heliozelidae dataset, suggesting that there is considerable conflict at this node. The GeneSortR topology was also recovered by the Scoutknife analysis of the original dataset with a support of 0.62 and by the original study28 with a UF bootstrap support of 65.1 and an SH-aLRT result of 72. Across our other Scoutknife simulations (Pseliastis+ Heliozela/Antispila/Antispilina/Holocacista/Coptodisca), was recovered with support ranging from 0.57 to 0.86. As a particularly short branch in all analyses, this could suggest that Scoutknife struggles to discern the topology when fewer genes have the capacity to resolve a node, or when incomplete lineage sorting increases substantially due to short branch lengths.\n\nAcross all 100 Simulation datasets, when the consensus value was lowered to a majority consensus tree, explicit agreement with the GeneSortR topology increased from an average of 92.85% to 98.62%, with explicit agreement values varying from 91.19% to 100%. This shows that, on average, in 5.77% of the nodes in the tree where GeneSortR displayed high confidence, Scoutknife instead assigned these nodes between 50 and 69% support. In accordance with this, Marczewski-Steinhaus similarity scores increased from an average of 0.93 to 0.99, with a variance from 0.85 – the aforementioned Simulation 20 – to 1 (Figure 4). Discounting the outlying Simulation 20, Marczewski-Steinhaus similarity scores vary from only 0.97 to 1. This further showcases the benefits of Scoutknife’s more conservative approach, making use of the diversity of data to give a more informed approximation of support from the gene trees without necessarily losing resolution at these key nodes.\n\nOur expectation was that as datasets became larger, they would become easier for Scoutknife to assess. However, instead, in both the Araneae and the simulation datasets, we found that Scoutknife was far more severely affected by per taxa dataset occupancy, rather than dataset size. In the simulation datasets, this takes the form of the simulation genes derived from Nothofagus, which is present in only 98 of the 1049 genes in each dataset. A consequence of this is that, in a truly representative 100-gene Scoutknife sample, a gene containing Nothofagus should be selected 9.3 times.\n\nAs an individual Scoutknife sample cannot select the same gene twice (although the same gene can be selected multiple times between samples), the probability of selecting any given gene can be modeled as a Hypergeometric distribution. This presents us with an understanding that only 60.52% of 100-gene Scoutknife samples will comprise at least 9 Nothofagus genes. On the other hand, there is a 99.99% chance that a 100-gene dataset contains at least one Nothofagus gene among the one hundred. However, in a 50-gene Scoutknife sample, there would be a 0.66% chance that 0 genes containing this taxon would be selected across the 1049. That means that across 100 50-gene Scoutknife samples, 1 sample of the 100 is likely to contain no representation of this taxon.\n\nIn this way, taxa with low gene occupancy have a far more notable effect on Scoutknife than reducing the number of genes, which evenly reduces the number of genes for all taxa in the dataset. This is not surprising as the principal assumption of the ScoutKnife procedure (Figure 1C) is that given genome-scale data works as a surrogate for unlimited data (Figure 1B). Accordingly, the power of ScoutKnife is driven by the availability of genome-scale data across the entire dataset and not just parts of it. The taxa with the lowest genomic representation set the ceiling for Scoutknife’s effectiveness, rather than those with reference genomes. All the 18 datasets used for this study were compiled before the reference genome revolution, which is still very recent16,17 and still restricted to only certain sections of the tree of life. Hence, for many taxa, genome-scale data at EBP minimum standards54 are still lacking. However, in the near future, the full potential of ScoutKnife can be brought to bear on these data. Our analyses already strongly indicate the potential of these methods in comparison to others through their conservativism in tree resolution and support values due to a higher susceptibility to the biological and methodological incongruence in the data.\n\nIn the meantime, the reduced power of ScoutKnife due to taxa with reduced genomic representation can be addressed by increasing the number of genes selected by a Scoutknife sample relative to poor taxon occupancy. This increases the absolute number of genes containing the low occupancy taxa in the dataset, though it will not affect the proportional representation of the low occupancy taxa. For example, to consider the Nothofagus earlier, a 200-gene Scoutknife dataset would increase the chance of observing 9 Nothofagus genes in any given Scoutknife sample from 60.52% to 99.84%. By doubling the size of the Scoutknife sample, a representative number of genes would be 18. However, simply increasing the raw representation of genes may aid Scoutknife resolution. This approach deviates from the naïve sampling strategy and introduces missingness as a selection parameter. On the other hand, this is often already done explicitly or implicitly as some genes can only be found in certain ingroups, for example, due to a gene duplication event, and so are generally excluded from these analyses in the dataset compilation step.\n\nAmong the tools available at the Scoutknife Github is a Hypergeometric distribution calculator designed with Scoutknife in mind, to help researchers understand the composition of their Scoutknife samples prior to analysis.\n\n\nConclusions\n\nSelection-based metrics have rightly dominated phylogenetic discussions for a great number of years, but in the era of big data, transitioning towards methods that make best use of the increased analytical power of whole genomes may be more prudent. Our results, and the Scoutknife methodology, show that, contrary to accepted wisdom, model violations and incongruence can be overcome by sheer density of data. What results is a more neutral look at phylogenetic relationships, rather than one biased by our own notions of what makes genes suitable for phylogenetics. A helpful side effect of this is an increase in computational efficiency: rather than assessing individual gene trees prior to multi-gene analysis, 100 smaller Scoutknife datasets assess the robusticity of a total dataset analysis or form the basis of a consensus tree. In many cases across our datasets, Scoutknife appears to recover the same relationships as before, but is also able to quantify our confidence in hypotheses of shared evolution efficiently and conservatively. In the future, this may be critical to a more holistic view of phylogeny. In addition, as models improve, and model incongruence becomes less and less of a concern, as a model-neutral methodology, Scoutknife’s ability to assess true biological incongruence will only improve, making it not only an exciting option for the present, but an even more effective one in the future.", "appendix": "Data availability\n\n\n\n• Source Data sets, 28 , 30 – 46 and their analysis within GeneSortR 19 were used to assess the efficacy of Scoutknife. All files used to assess the efficacy of Scoutknife can be found reproduced in our underlying data link (below). Further information on the Source datasets can also be found in the supplemental data for Koch et al. (2021). 19\n\n\n\n• Both our real and simulated data analyses are available at DataDryad, along with copies of individual gene fasta files from Source Data sets 28 , 30 – 46 and the 250 most informative gene trees from Koch et al. (2021) 19 that were used to benchmark Scoutknife’s performance (https://datadryad.org/stash/dataset/doi:10.5061/dryad.sxksn0383).\n\n\nAcknowledgements\n\nThe authors would like to recognise Tomas Berger for developing Scoutknifette, making the large number of MPI submissions necessary for the project more manageable.\n\n\nReferences\n\nFleming JF, Valero-Gracia A, Struck TH: Identifying and addressing methodological incongruence in phylogenomics: A review. Evol. Appl. 2023; 16: 1087–1104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolfe KH, Li W-H: Molecular evolution meets the genomics revolution. Nat. Genet. 2003; 33(3): 255–265. PubMed Abstract \| Publisher Full Text\n\nGee H: Ending incongruence. Nature. 2003 2003/10; 425(6960): 782. Publisher Full Text\n\nBortoluzzi C, Wright CJ, Lee S, et al.: Lepidoptera genomics based on 88 chromosomal reference sequences informs population genetic parameters for conservation. bioRxiv. 2023:2023.04.14.536868.\n\nChallis R, Kumar S, Sotero-Caio C, et al.: Genomes on a Tree (GoaT): A versatile, scalable search engine for genomic and sequencing project metadata across the eukaryotic tree of life. Wellcome Open Res. 2023; 8(24): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMirarab S, Nakhleh L, Warnow T: Multispecies Coalescent: Theory and Applications in Phylogenetics. Annu. Rev. Ecol. Evol. Syst. 2021 2021/11/02; 52(1): 247–268. Publisher Full Text\n\nMirarab S, Bayzid MS, Warnow T: Evaluating Summary Methods for Multilocus Species Tree Estimation in the Presence of Incomplete Lineage Sorting. Syst. Biol. 2014 2014/08/26; 65(3): 366–380. PubMed Abstract \| Publisher Full Text\n\nErixon P, Svennblad B, Britton T, et al.: Reliability of Bayesian posterior probabilities and bootstrap frequencies in phylogenetics. Syst. Biol. 2003; 52(5): 665–673. PubMed Abstract \| Publisher Full Text\n\nAnisimova M, Gil M, Dufayard J-F, et al.: Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. Syst. Biol. 2011; 60(5): 685–699. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnisimova M, Gascuel O: Approximate Likelihood-Ratio Test for Branches: A Fast, Accurate, and Powerful Alternative. Syst. Biol. 2006 2006/08/01; 55(4): 539–552. PubMed Abstract \| Publisher Full Text\n\nFelsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39(4): 783–791. PubMed Abstract \| Publisher Full Text\n\nSiddall ME: Another monophyly index: revisiting the jackknife. Cladistics. 1995; 11(1): 33–56. PubMed Abstract \| Publisher Full Text\n\nSoltis PS, Soltis DE: Applying the bootstrap in phylogeny reconstruction. Stat. Sci. 2003; 18: 256–267. Publisher Full Text\n\nSwofford DL: Phylogenic inference. Molecular systematic. 1996.\n\nPaez S, Kraus RH, Shapiro B, et al.: Reference genomes for conservation. Science. 2022; 377(6604): 364–366. Publisher Full Text\n\nLewin HA, Richards S, Lieberman Aiden E, et al.: The earth BioGenome project 2020: Starting the clock. National Acad Sciences. 2022; 119: e2115635118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEbenezer TE, Muigai AW, Nouala S, et al.: Africa: sequence 100,000 species to safeguard biodiversity. Nature. 2022; 603(7901): 388–392. PubMed Abstract \| Publisher Full Text\n\nLozano-Fernandez J: A Practical Guide to Design and Assess a Phylogenomic Study. Genome Biol. Evol. 2022; 14(9): evac129. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMongiardino KN: Phylogenomic subsampling and the search for phylogenetically reliable loci. Mol. Biol. Evol. 2021; 38(9): 4025–4038. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards SV: Phylogenomic subsampling: a brief review. Zool. Scr. 2016; 45: 63–74. Publisher Full Text\n\nRabiee M, Sayyari E, Mirarab S: Multi-allele species reconstruction using ASTRAL. Mol. Phylogenet. Evol. 2019; 130: 286–296. PubMed Abstract \| Publisher Full Text\n\nHillis DM, Moritz C, Mable BK: Molecular systematics. Sinauer; 1996.\n\nCreevey C, Weeks N: ChrisCreevey/catsequences: Version 1.3. Zenodo. 2021.\n\nNguyen L-T, Schmidt HA, von Haeseler A , et al.: IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015; 32(1): 268–274. PubMed Abstract \| Publisher Full Text\n\nKalyaanamoorthy S, Minh BQ, Wong TK, et al.: ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods. 2017; 14(6): 587–589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLartillot N, Lepage T, Blanquart S: PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics. 2009 2009/06/17; 25(17): 2286–2288. PubMed Abstract \| Publisher Full Text\n\nSukumaran J, Holder MT: DendroPy: a Python library for phylogenetic computing. Bioinformatics. 2010; 26(12): 1569–1571. PubMed Abstract \| Publisher Full Text\n\nMilla L, Moussalli A, Wilcox SA, et al.: Phylotranscriptomics resolves phylogeny of the Heliozelidae (Adeloidea: Lepidoptera) and suggests a Late Cretaceous origin in Australia. Syst. Entomol. 2020; 45(1): 128–143. Publisher Full Text\n\nRdpack R, Rcpp L: Package ‘Quartet’. Adv. Appl. Math. 2019; 7: 309–343.\n\nHughes LC, Ortí G, Huang Y, et al.: Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data. Proc. Natl. Acad. Sci. 2018; 115(24): 6249–6254. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández R, Kallal RJ, Dimitrov D, et al.: Phylogenomics, diversification dynamics, and comparative transcriptomics across the spider tree of life. Curr. Biol. 2018; 28(9): 1489–1497.e5. Publisher Full Text\n\nSteenwyk JL, Shen X-X, Lind AL, et al.: A robust phylogenomic time tree for biotechnologically and medically important fungi in the genera Aspergillus and Penicillium. MBio. 2019; 10(4): e00925–e00919. Publisher Full Text\n\nEvangelista DA, Wipfler B, Béthoux O, et al.: An integrative phylogenomic approach illuminates the evolutionary history of cockroaches and termites (Blattodea). Proc. R. Soc. B. 1895; 2019(286): 20182076.\n\nMongiardino Koch N, Thompson JR: A total-evidence dated phylogeny of Echinoidea combining phylogenomic and paleontological data. Syst. Biol. 2021; 70(3): 421–439. Publisher Full Text\n\nIrisarri I, Baurain D, Brinkmann H, et al.: Phylotranscriptomic consolidation of the jawed vertebrate timetree. Nat. Ecol. Evol. 2017; 1(9): 1370–1378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson KP, Dietrich CH, Friedrich F, et al.: Phylogenomics and the evolution of hemipteroid insects. Proc. Natl. Acad. Sci. 2018; 115(50): 12775–12780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMisof B, Liu S, Meusemann K, et al.: Phylogenomics resolves the timing and pattern of insect evolution. Science. 2014; 346(6210): 763–767. PubMed Abstract \| Publisher Full Text\n\nPeters RS, Krogmann L, Mayer C, et al.: Evolutionary history of the Hymenoptera. Curr. Biol. 2017; 27(7): 1013–1018. Publisher Full Text\n\nKawahara AY, Plotkin D, Espeland M, et al.: Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths. Proc. Natl. Acad. Sci. 2019; 116(45): 22657–22663. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen H, Jin D, Shu J-P, et al.: Large-scale phylogenomic analysis resolves a backbone phylogeny in ferns. GigaScience. 2018; 7(2): gix116. Publisher Full Text\n\nFernández R, Edgecombe GD, Giribet G: Exploring phylogenetic relationships within Myriapoda and the effects of matrix composition and occupancy on phylogenomic reconstruction. Syst. Biol. 2016; 65(5): 871–889. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández R, Sharma PP, Tourinho AL, et al.: The Opiliones tree of life: shedding light on harvestmen relationships through transcriptomics. Proc. R. Soc. B Biol. Sci. 2017; 284(1849): 20162340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimon S, Letsch H, Bank S, et al.: Old World and New World Phasmatodea: phylogenomics resolve the evolutionary history of stick and leaf insects. Front. Ecol. Evol. 2019; 7: 345. Publisher Full Text\n\nBenavides LR, Cosgrove JG, Harvey MS, et al.: Phylogenomic interrogation resolves the backbone of the Pseudoscorpiones tree of life. Mol. Phylogenet. Evol. 2019; 139: 106509. Publisher Full Text\n\nShen X-X, Opulente DA, Kominek J, et al.: Tempo and mode of genome evolution in the budding yeast subphylum. Cell. 2018; 175(6): 1533–1545.e20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharma PP, Baker CM, Cosgrove JG, et al.: A revised dated phylogeny of scorpions: phylogenomic support for ancient divergence of the temperate Gondwanan family Bothriuridae. Mol. Phylogenet. Evol. 2018; 122: 37–45. PubMed Abstract \| Publisher Full Text\n\nLy-Trong N, Naser-Khdour S, Lanfear R, et al.: Alisim: A fast and versatile phylogenetic sequence simulator for the genomic era. Mol. Biol. Evol. 2022; 39(5): msac092. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinh BQ, Schmidt HA, Chernomor O, et al.: IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 2020; 37(5): 1530–1534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith MR: Bayesian and parsimony approaches reconstruct informative trees from simulated morphological datasets. Biol. Lett. 2019; 15(2): 20180632. Publisher Full Text\n\nEstabrook GF, McMorris F, Meacham CA: Comparison of undirected phylogenetic trees based on subtrees of four evolutionary units. Syst. Zool. 1985; 34(2): 193–200. Publisher Full Text\n\nDay WHE: Analysis of Quartet Dissimilarity Measures Between Undirected Phylogenetic Trees. Syst. Biol. 1986; 35(3): 325–333. Publisher Full Text\n\nSteel MA, Penny D: Distributions of tree comparison metrics—some new results. Syst. Biol. 1993; 42(2): 126–141.\n\nSimmons MP, Goloboff PA, Stöver BC, et al.: Quantification of congruence among gene trees with polytomies using overall success of resolution for phylogenomic coalescent analyses. Cladistics.\n\nProject EB. Reference Source" }	[ { "id": "227172", "date": "29 Dec 2023", "name": "Paul Zaharias", "expertise": [ "Reviewer Expertise Phylogenetics", "systematics 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\nThis article introduces “Scoutknife”, a software designed for gene jackknifing in phylogenomic datasets. The authors evaluate their approach with numerous empirical and simulated datasets and using a range of similarity metrics. They conclude that Scoutknife is a reliable estimator of the robustness of phylogenetic hypotheses. I find the article well-written and easy to follow, and that “Scoutknife” could be a useful user-friendly software for phylogenomists. I only have two major comments, followed by some minor comments hereafter. First, the method is not new (contrarily to what can be read in the Introduction, paragraph 6); the procedure of gene jackknifing exists since at least 15 years ago [1,2]. As such, I would like to see in the Introduction a paragraph which would be a summary of the literature on the use of gene jackknifing, its assessed qualities, and drawbacks. This is however, the first time (to my knowledge) that a user-friendly software is suggested, and that there is a proper benchmarking of it, and the authors should emphasize on those aspects. Second, as the author point out in the discussion, one drawback of gene jackknifing (as it is with site-based jackknifing) is the problem of poor taxon occupancy, unfortunately a common problem in phylogenomic datasets. To address that, the authors wrote a “Hypergeometric distribution calculator” script to help the users better understand the taxon occupancy composition prior to analysis. I think this script should be automated and included as an option in the main “Scoutknife” approach as follow:\nThe Scoutknife script automatically detects the number of genes in the folder and also automatically detects how many of those genes contain the least represented taxa. Then, Scoutknife calculates how many genes should be sampled at least so that there is >99.9% chances that the least represented taxon appears in at least one gene. This value could be considered the default minimal value of gene sampling for the user to guarantee the presence of all taxa in the subsets.\nMaking the use of this “Taxon Probability Calculator\" more visible and user-friendly would be a great plus and a great input to the more arbitrary “100 genes” strategy fixed by the authors and would show an adaptiveness of the approach to each dataset.\nSome minor comments: Everywhere: “Pseudosampling” in Fig.1 caption is not spelled the same way as other places in the text (“Pseudo sampling”). Might I suggest as well to add a hyphen after all “pseudo” as in “pseudo-sampling”, “pseudo-samples” and “pseudo-replicates”? Discussion, penultimate paragraph: please expand EBP to Earth BioGenome Project Last comment, which is more of a curiosity : is there any particular reason you chose the synonym \"robusticity' instead of the more traditional \"robustness\" present in the literature ?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11957", "date": "10 Jul 2024", "name": "James Fleming", "role": "Author Response", "response": "We would like to thank the reviewer for their positive response to the paper, and we found the comments below incredibly useful in improving the manuscript (which we are currently uploading). To address their concerns one-by-one below: ================== First, the method is not new (contrarily to what can be read in the Introduction, paragraph 6); the procedure of gene jackknifing exists since at least 15 years ago [1,2]. As such, I would like to see in the Introduction a paragraph which would be a summary of the literature on the use of gene jackknifing, its assessed qualities, and drawbacks. This is however, the first time (to my knowledge) that a user-friendly software is suggested, and that there is a proper benchmarking of it, and the authors should emphasize on those aspects. ----- Scoutknife is not quite the same as gene jacknifing. The two are similar but quite distinct in their mathematical underpinnings and philosophical approach - in particular, jackknifing as typically implemented preserves the relative order of genes in the genome, and is also used predominantly to assess the effect of order on the resultant dataset, following initial phylogenetic tree construction. Scoutknife is much more similar to a classical bootstrapping approach, where the creation of alignment sets does not respect gene order. However, the reviewer is totally correct that these are relatively minor distinctions between the two approaches that were not clearly delineated in our initial manuscript, and that leaving out discussion of gene jacknifing is an important oversight. We have revised the initial introduction section to discuss jacknifing, and the differences between scoutknife and jacknife in more detail. We have also been more computationally explicit about the intentions of Scoutknife, with a new script, Scoutknife_Consensus.pl, being included in the GitHub, to allow users to easily generate consensus trees within the package itself, rather than relying on alternative programs as we have done in the manuscript itself. ==================== Second, as the author point out in the discussion, one drawback of gene jackknifing (as it is with site-based jackknifing) is the problem of poor taxon occupancy, unfortunately a common problem in phylogenomic datasets. To address that, the authors wrote a “Hypergeometric distribution calculator” script to help the users better understand the taxon occupancy composition prior to analysis. I think this script should be automated and included as an option in the main “Scoutknife” approach as follow: The Scoutknife script automatically detects the number of genes in the folder and also automatically detects how many of those genes contain the least represented taxa. Then, Scoutknife calculates how many genes should be sampled at least so that there is >99.9% chances that the least represented taxon appears in at least one gene. This value could be considered the default minimal value of gene sampling for the user to guarantee the presence of all taxa in the subsets. Making the use of this “Taxon Probability Calculator\" more visible and user-friendly would be a great plus and a great input to the more arbitrary “100 genes” strategy fixed by the authors and would show an adaptiveness of the approach to each dataset. ----- That is a great idea! We have included an \"--auto\" flag inside Scoutknife that works as the reviewer describes. ====================== Some minor comments: ------ We have made revisions based on all minor comments. For the reviewer's curiosity, it was mostly the first author writing naturally - I'm not sure I'd put too much thought into that particular terminology usage, but you are totally correct to point it out. I have adjusted the terminology there appropriately." } ] }, { "id": "261535", "date": "06 May 2024", "name": "Xianzhao Kan", "expertise": [ "Reviewer Expertise Bioinformatics", "Genomics", "and Molecular evolution." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript, \"Scoutknife: A Naïve, Whole-Genome Informed Phylogenetic Robustness Metric\", introduces a novel tool, Scoutknife. In this method, the authors can potentially abandon the notion of pseudo-sampling and instead randomly sample small subsets of genes from the thousands of genes in their analyses. Furthermore, the authors validated the effectiveness of Scoutknife by applying it to 18 previously published datasets and 100 simulation studies. I believe that the development of this method and software will facilitate deeper research in phylogenetics.\n\nThe following weaknesses should be addressed: 1. In the Abstract section, the term \"robusticity\" is used frequently, but I think it is not a standard statistical term. \"Robustness\" would be more suitable. 2. In the Conclusions of the Abstract, \"phylogenetic hypotheses\" should be changed to \"phylogenetic hypothesis\" because it refers to a singular hypothesis. 3. In the paragraph 4 of Introduction, the sentence \"Currently, phylogenetics has adopted a conservative stance, selecting genes that fit well within our models of evolution – this has the benefit of evading artifactual topologies, but may well be presenting us with hypotheses of evolution that are preselected according to our own biases, or incorrectly causing us to adopt great confidence in hypotheses that are not as well supported by the data as it first appears.\" is quite long and could be split into multiple sentences for clarity. 4. In the paragraph 6 of Introduction, the phrase \"true replicate samples\" may be confusing. It would be helpful to clarify the difference between \"true replicates\" and \"artificial pseudo-replicates\". 5. In the section on Scoutknife and per-taxon gene occupancy, the following sentence is unclear: \"genome-scale data works as a surrogate for unlimited data\". The authors should clarify the relationship between genome-scale data and the concept of unlimited data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "11958", "date": "10 Jul 2024", "name": "James Fleming", "role": "Author Response", "response": "We thank the reviewer for their consideration of our manuscript, and have attempted to clarify and address our text. We would like to especially thank them for point out the lack of clarity in points 4 and 5: these are quite critical concepts to understanding the wider manuscript, so it is important they are explained as clearly as possible. In order to address this, we have greatly revised the initial introduction section in order to hopefully explain the concepts and reasoning behind Scoutknife more clearly." } ] }, { "id": "195414", "date": "25 May 2024", "name": "Anthony K. Redmond", "expertise": [ "Reviewer Expertise Phylogenomics", "Genome Evolution", "Gene and Genome Duplication", "Immunology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well written and presented study by Fleming and colleagues provides an important and much needed alternative measure of robustness for phylogenetic inference based on gene-wise jackknife resampling. The logical and practical ideas behind, and advantages of using, their approach are clearly explained and exemplified in comparison to the traditional site-wise bootstrapping approach.\nWhile I must admit that the study has not entirely convinced me that 'model violations and incongruence can be overcome by sheer density of data' (and this may be a bias of my own), I am nonetheless very pleased with the conservative and nuanced results provided by the new Scoutknife approach as compared to standard bootstrap. I believe the Scoutknife approach has great potential within the field and am happy to recommend the manuscript without revision. I am excited to see how the approach holds up to extreme lineage-specific violations on the modelling and orthology fronts.\nAs a very minor point and optional point, I believe there may be additional studies worthy of discussion here that support some of the arguements made in the text. For example, Simmons et al. 2019 [ ref 1 ] previously proposed the use of gene-wise bootstrap for summary coalescent species tree methods, while gene wise bootstrapping has been used in other studies in the past (e.g. Simion et al 2017 [ ref 2 ]), and off the top of my head has been suggested to be conservative in at least Philippe et al 2019 [ ref 3 ].\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-945
https://f1000research.com/articles/12-324/v1	23 Mar 23	{ "type": "Data Note", "title": "The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence", "authors": [ "Riham Ayoubi", "Walaa Alshafie", "Irina Shlaifer", "Kathleen Southern", "Peter S. McPherson", "Carl Laflamme", "NeuroSGC/YCharOS/EDDU collaborative group", "Riham Ayoubi", "Walaa Alshafie", "Irina Shlaifer", "Kathleen Southern", "Peter S. McPherson" ], "abstract": "Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.", "keywords": [ "Uniprot ID Q13501", "SQSTM1", "Sequestosome-1", "antibody characterization", "antibody validation", "Western blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nSequestosome-1, alternatively known as p62, is an adaptor protein required for selective autophagy and proteasomal degradation.1–3 Delivering polyubiquitinated proteins to the autophagosome or proteasome, Sequestosome-1/p62 plays a key role in the degradation of aggregate prone proteins.1\n\nSQSTM1 gene mutations may act as a potential threat by causing altered autophagy, resulting in pathogenic protein aggregation and the development of a variety of neurodegenerative diseases, including ALS and FTD.4 Furthermore, SQSTM1 mutations have been identified in patients with ALS and FTD.5 Serving as a signalling hub for neurodegenerative pathways, Sequestosome-1/p62 poses as a prospective therapeutic target in the treatment of neurodegenerative diseases.6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.\n\nHere, we compared the performance of a range of commercially available antibodies for Sequestosome-1 and validated high-performing antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Sequestosome-1 properties and function.\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.7–9 To identify a cell line that expresses adequate levels of Sequestosome-1 protein to provide sufficient signal to noise we examined public proteomics databases, namely PAXdb (RRID:SCR_018910)10 and DepMap (RRID:SCR_017655).11 U2OS was identified as a suitable cell line and thus U2OS was modified with CRISPR/Cas9 to knockout the corresponding SQSTM1 gene (Table 1).\n\nFor Western blot experiments, we resolved proteins from WT and SQSTM1 KO cell extracts and probed them side-by-side with all antibodies in parallel8,9 (Figure 1).\n\nLysates of U2OS (WT and SQSTM1 KO) were prepared and 25 μg of protein were processed for Western blot with the indicated Sequestosome-1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies MA5-32835, 66184-1-Ig and 18420-1-AP, which were titrated to 1/200, 1/1000 and 1/1000, respectively, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: GTX629890* at 1/1000; GTX629888* at 1/1000; GTX100685 at 1/1000; 701510 at 1/1000; 710539 at 1/200; MA5-32835* at 1/200; 66184-1-Ig* at 1/1000; 18420-1-AP at 1/1000; 55274-1-AP at 1/1000; NBP2-23490* at 1/1000; MAB80281** at 1/1000; MAB8028* at 1/1000; MAB8028R at 1/1000; ab109012 at 1/10000; ab56416* at 1/1000; ab207305 at 1/1000; ab240635 at 1/1000. Predicted band size: ~62 kDa. = monoclonal antibody, = recombinant antibody.\n\nFor immunoprecipitation experiments, we used the antibodies to immunopurify Sequestosome-1 from U2OS cell extracts. The performance of each antibody was evaluated by detecting the Sequestosome-1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates8,9 (Figure 2).\n\nU2OS lysates were prepared, and IP was performed using 1.0 μg of the indicated Sequestosome-1 antibodies pre-coupled to protein G or protein A Sepharose beads. Samples were washed and processed for Western blot with the indicated Sequestosome-1 antibody. For Western blot, MAB80281* was used at 1/3000, 66184-1-lg* at 1/3000, ab56416* at 1/5000, ab207305** at 1/10000 and GTX629890* at 1/5000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM= 10% starting material; UB=10% unbound fraction; IP=immunoprecipitate, HC= antibody heavy chain. = monoclonal antibody, = recombinant antibody.\n\nFor immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.12 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).\n\nU2OS WT and SQSTM1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated Sequestosome-1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody. Acquisition of the green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of red (grayscale images) channel are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies MA5-32835, 18420-1-AP and NBP2-23490, which were titrated to 1/2000, 1/300 and 1/1000, respectively, as the signals were too strong when following the supplier’s recommendations. When the concentration was not indicated by the supplier, we tested antibodies at 1/500 and 1/1000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: GTX629890 at 1/1000; GTX629888* at 1/1000; GTX100685 at 1/700; 701510 at 1/500; 710539 at 1/500; MA5-32835* at 1/2000; 66184-1-Ig* at 1/300; 18420-1-AP at 1/300; 55274-1-AP at 1/1300; NBP2-23490* at 1/1000; MAB80281** at 1/500; MAB8028* at 1/500; MAB8028R at 1/500; ab109012 at 1/500; ab56416* at 1/1000; ab207305 at 1/200; ab240635 at 1/500. Bars = 10 μm. = monoclonal antibody, = recombinant antibody.\n\nIn conclusion, we have screened Sequestosome-1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and characterized several high-quality antibodies under our standardized experimental conditions. The underlying data can be found on Zenodo.13,14\n\n\nMethods\n\nAll Sequestosome-1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.15 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429).\n\n = monoclonal antibody.\n\n** = recombinant antibody.\n\nSQSTM1 KO clone was generated in Cas9-expressing U2OS cell line7 with low passage cells using an open-access protocol available on Zenodo.org: https://zenodo.org/record/3875777#.ZA9VdC-96Tf. Two guide RNAs were used to introduce a STOP codon in the SQSTM1 gene (sequence guide 1: CCACCGCCCACCGUGUGCUC, sequence guide 2: AUGCGAGCUUGGUGUGCCCC).\n\nBoth U2OS WT and SQSTM1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.16 Cells were cultured in DMEM high glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201).\n\nWestern blots were performed as described in our standard operating procedure.17 U2OS WT and SQSTM1 KO were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 x g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used.\n\nWestern blots were performed with large 4-15% polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining, which is scanned to show them together with individual Western blots. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signaling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with regular ECL (cat. number 32106) or super signal West Femto (cat. number 34096) from Thermo Fisher Scientific prior to detection with the HyBlot CL autoradiography films from Denville (cat. number 1159T41).\n\nImmunoprecipitation was performed as described in our standard operating procedure.18 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) with 0.01% triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μl of protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by several washes to remove unbound antibodies.\n\nU2OS WT were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor (MilliporeSigma, cat. number P8340). Lysates were rocked 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C. One ml aliquots at 1.0 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hours at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of HEPES lysis buffer and processed for SDS-PAGE and Western blot on a 4-15% polyacrylamide gels as described above.\n\nImmunofluorescence was performed as described in our standard operating procedure.8,9,12 U2OS WT and SQSTM1 KO were labelled with a green and a deep red fluorescence dye from Abcam (cat. number ab176735 and ab176736), respectively. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37°C, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in PBS for 15 min at room temperature and then washed three times with PBS. Cells were permeabilized in PBS with 0.1% triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary Sequestosome-1 antibodies overnight at 4°C. Cells were then washed 3 x 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature. Cells were washed 3 x 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).\n\nImaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40x oil objective (NA = 1.40). Analysis was done using ImageJ (RRID:SCR_003070). All cell images represent a single focal plane. Figures were assembled with Adobe Photoshop (version 24.2.1) (RRID:SCR_014199) to adjust contrast and apply 1-pixel Gaussian blur, and then they were assembled with Adobe Illustrator (version 27.3.1) (RRID:SCR_010279).", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Sequestosome-1, https://doi.org/10.5281/zenodo.4731001. 13\n\nZenodo: Dataset for the Sequestosome-1 antibody screening study, https://doi.org/10.5281/zenodo.7709902. 14\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, Irina Shlaifer and Kathleen Southern.\n\nAn earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.4818440).\n\n\nReferences\n\nTurco E, Savova A, Gere F, et al.: Reconstitution defines the roles of p62, NBR1 and TAX1BP1 in ubiquitin condensate formation and autophagy initiation. Nat. Commun. 2021; 12(1): 5212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBjørkøy G, Lamark T, Brech A, et al.: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 2005; 171(4): 603–614. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClausen TH, Lamark T, Isakson P, et al.: p62/SQSTM1 and ALFY interact to facilitate the formation of p62 bodies/ALIS and their degradation by autophagy. Autophagy. 2010; 6(3): 330–344. PubMed Abstract \| Publisher Full Text\n\nRea SL, Majcher V, Searle MS, et al.: SQSTM1 mutations--bridging Paget disease of bone and ALS/FTLD. Exp. Cell Res. 2014; 325(1): 27–37. PubMed Abstract \| Publisher Full Text\n\nLe Ber I, Camuzat A, Guerreiro R, et al.: SQSTM1 mutations in French patients with frontotemporal dementia or frontotemporal dementia with amyotrophic lateral sclerosis. JAMA Neurol. 2013; 70(11): 1403–1410. PubMed Abstract \| Publisher Full Text\n\nMa S, Attarwala IY, Xie XQ: SQSTM1/p62: A Potential Target for Neurodegenerative Disease. ACS Chem. Neurosci. 2019; 10(5): 2094–2114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 2023(12): 172.\n\nWang M, Herrmann CJ, Simonovic M, et al.: Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines. Proteomics. 2015; 15(18): 3163–3168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNusinow DP, Szpyt J, Ghandi M, et al.: Quantitative Proteomics of the Cancer Cell Line Encyclopedia. Cell. 2020; 180(2): 387–402. e16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nAyoubi R, Alshafie W, Shlaifer I, et al.: Antibody Characterization Report for Sequestosome-1. [Dataset]. Zenodo 2021.\n\nLaflamme C: Dataset for the Sequestosome-1 antibody screening study. [Dataset]. Zenodo. 2023.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021." }	[ { "id": "247622", "date": "21 Feb 2024", "name": "Yasukazu Takanezawa", "expertise": [ "Reviewer Expertise As a specialist in toxicology", "particularly focused on the interplay between autophagy and heavy metals." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 delves into the intricacies of Western blot, immunoprecipitation, and immunofluorescence techniques utilizing commercially available antibodies targeting p62, particularly in the context of neurodegenerative disease pathogenesis. This paper provides valuable insights into the application of p62 antibodies. Additionally, incorporating co-staining with LC3 would enhance its informativeness. Moreover, exploring the characterization of anti-p62 antibodies from other companies would be advantageous.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "247595", "date": "21 Feb 2024", "name": "Guoqiang Xu", "expertise": [ "Reviewer Expertise biochemistry", "pharmacology", "cancer biology", "proteomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 used the WT and Sequestosome-1 knockout U2OS cells to evaluate the quality of the anti-Sequestosome-1 antibodies obtained from different companies in three different applications, i.e. WB, IP, and IF. The results are very useful for other scientists to choose the right antibody for the specific application.\nIn the paper, WT and Sequestosome-1 knockout U2OS cells were used for the experiment. Additional cell lines, especially for different cancer cell lines and neuronal cell lines should also be tested since the antibodies will be most probably used in these cell lines in real situations. In addition, different cell lines have different levels of protein expression and post-translational modifications, which are also frequently affecting the experimental results.\nFor Figure 2, why do only two blots have bands for Heavy chains (HC)? An explanation should be provided in the figure legend or the text for better understanding.\nFor Figure 3, it is recommended that the authors provide the green, red, and contrast images for each antibody for better visualization and assessment.\nA conclusion should be provided at the end of the paper to show the quality and application of the different antibodies tested in this paper. A comparison among different antibodies is very valuable for other scientists. In addition, it will be useful to discuss the possible advantages and disadvantages of this work.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11967", "date": "10 Jul 2024", "name": "Kathleen Southern", "role": "Author Response", "response": "Thank you Guoqiang Xu for taking the time to thoroughly review this Data Note, we appreciate your constructive feedback and hope our responses relieve any concerns or questions you previously had. As such, a new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below. In the paper, WT and Sequestosome-1 knockout U2OS cells were used for the experiment. Additional cell lines, especially for different cancer cell lines and neuronal cell lines should also be tested since the antibodies will be most probably used in these cell lines in real situations. In addition, different cell lines have different levels of protein expression and post-translational modifications, which are also frequently affecting the experimental results. Response: We understand how the cell line selection process was not well understood, and we have edited the first paragraph of the Results and discussion section to clarify this part of our protocol. From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 6.7 log2 (TPM+1), which is significantly above the minimum threshold level, 2.5 log2 (TPM+1), previously established. A limitation of the orthogonal knockout (KO) based approach to test antibodies is that only one cell line (WT and KO) is used to evaluate the performance of the antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using WT and an equivalent KO cell line, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Protocol exchange and (DOI:10.21203/rs.3.pex-2607/v1). This reference has been included in the new version. For Figure 2, why do only two blots have bands for Heavy chains (HC)? An explanation should be provided in the figure legend or the text for better understanding. Response: In the protocol exchange paper, previously referred to, which outlines the antibody characterization procedure followed in this article, and is now referenced in the new version of the article. Within the protocol, we have a troubleshooting section with proposed solutions to prevent the heavy chains from appearing. It can be found in the IP Procedure section 2E). Although we have tried to optimize the workflow, we cannot control whether the heavy chain bands appear and show the IP results as we do with all the other antibodies tested. For Figure 3, it is recommended that the authors provide the green, red, and contrast images for each antibody for better visualization and assessment. Response: This underlying data is provided in the Dataset, found in the Data availability section. You can also use the following link to be redirected to the Dataset, https://doi.org/10.5281/zenodo.7709902. A conclusion should be provided at the end of the paper to show the quality and application of the different antibodies tested in this paper. A comparison among different antibodies is very valuable for other scientists. In addition, it will be useful to discuss the possible advantages and disadvantages of this work. Response: The authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1). Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded. That being said, we understand how this aspect of the YCharOS initiative was not clearly defined and in the newly submitted version we have included two paragraphs to the end of the introduction that will provide an understanding as to why we do not analyze the results, preventing further misinterpretation." } ] } ]	1	https://f1000research.com/articles/12-324
https://f1000research.com/articles/10-1009/v1	05 Oct 21	{ "type": "Review", "title": "Evidence of mitochondria origin of SARS-CoV-2 double-membrane vesicles: a review.", "authors": [ "Pavel Montes de Oca-B" ], "abstract": "Coronavirus Disease-19 (COVID-19) pandemic is caused by the coronavirus SARS-CoV-2 that has infected in a year more than 200 million people and has killed almost 4.5 million people worldwide. This infection affects mainly certain groups of people that have high susceptibility to present severe COVID-19 due to comorbidities. Moreover, the long-COVID-19 comprises a series of symptoms that may remain in some patients for months after infection that further compromises health of individuals. Therefore, this pandemic poses a serious emergency worldwide. Thus, since this pandemic is profoundly affecting economic and social life of societies, a deeper understanding of SARS-CoV-2 infection cycle could help to envisage novel therapeutic alternatives that limit or stop COVID-19. Several recent findings have unexpectedly found that mitochondria play a critical role in SARS-CoV-2 cell infection. Indeed, it has been suggested that this organelle could be the origin of its replication niches, the double membrane vesicles (DMV), as its been observed with another virus. In this regard, mitochondria derived vesicles (MDV), involved in mitochondria quality control, were discovered more than 10 years ago and interestingly there is a population characterized by a double membrane. MDV shedding is induced by mitochondrial stress and it has a fast assembly dynamic, reason that perhaps has precluded their identification in electron microscopy or tomography studies. These and other features of MDV together with recent SARS-CoV-2 protein interactome with the host and other findings linking SARS-CoV-2 to mitochondria, support that these vesicles are the precursors of SARS-CoV-2 induced DMV. In this work, the celular, molecular, phenotypical and biochemical evidence that supports this hypothesis is reviewed and integrated into the current model of SARS-CoV-2 cell infection. In this scheme, some relevant questions are raised as pending topics for research that would help in the near future to test this hypothesis. The intention (abstract truncated).", "keywords": [ "SARS CoV-2", "DMV", "MDV", "mitochondria", "caveolae", "COVID" ], "content": "Background\n\nThe coronavirus disease-19 (COVID-19) pandemic is caused by the positive-sense single-stranded RNA coronavirus, SARS-CoV-2, that has infected, in a year, more than 200 million people and has killed almost 4.5 million people worldwide1 since it has no definitive and effective treatment until today. This infection affects mainly certain groups of people that have high susceptibility to present severe COVID-19 due to comorbidities that include cardiovascular disease, chronic kidney, respiratory or liver disease, severe obesity, or hypertension among others. In these patients, the cytokine storm induced by the virus poses a serious death risk for these patients due to the systemic inflammation and multiorgan failure2–6. Moreover, the so-called long COVID-19 comprises a series of symptoms that may remain in some patients for months after infection that further compromises their health, even after non-severe COVID-197. Despite huge efforts to stop infections and deaths worldwide, only a few treatments have been demonstrated to ameliorate severe COVID-19, and different vaccine strategies are currently under investigation in clinical phase III and/or IV trials. Therefore, in this scenario, a deeper understanding of the cellular mechanisms exploited by SARS-CoV-2 for cell infection could undoubtedly provide new unforeseen strategies to tackle this pandemic.\n\n\nThe SARS-CoV-2 replication organelles and the unresolved origin of double-membrane vesicles\n\nSeveral recent reports have shown that mitochondria play a relevant role during SARS-CoV-2 infection8–10. These findings and previously published results of SARS-CoV-2, SARS-CoV and other coronavirus biology allow us to hypothesize that mitochondria could be responsible for the assembly of double-membrane vesicles (DMV). These are membrane modifications induced by SARS-CoV-2 and it is where viralRNA (vRNA) replication occurs in the infected cell, that are believed to be derived from the endoplasmic reticulum (ER) or other mechanisms, such as autophagy11–13. However, some published literature supports that double-membrane mitochondria-derived vesicles (MDV), discovered some years ago14, could be the precursors or relatives of DMV. This hypothesis of mitochondria role in DMV assembly and the involvement of MDV has been suggested previously8,15,16. Indeed, specialized replication organelles (RO) at mitochondria outer membrane (MOM) have been observed in FHV17, whereas HIV RNA is known to locate in the mitochondria18. Here, a brief review of the evidence that supports this notion is presented and integrated into the current model19, with the intention to provide a novel framework that could open possibilities to tackle the SARS-CoV-2 pandemic.\n\nDMV along with other membrane modifications are part of the RO induced by SARS-CoV-2 that also includes convoluted membranes (CM), zippered ER (zER), vesicle packets (VP), and double-membrane spherules (DMS)19 (Figure 1). RO are induced by SARS-CoV-2 in infected cells, as a variety of RO are induced by viruses including other nidovirus and picornavirus among others11,13. DMV assembly is induced by viral proteins but seems to also require other viral or host factors because cell plasmid transfection of transmembrane-containing non-structural proteins (nsp) 3-, 4-, and 6-induced membrane arrangements that resemble DMV but with smaller size20. These nsp are part of the complex involved in vRNA replication, together with nsp12, the RNA-dependent RNA polymerase, and other nsp. Consistently, nsp4 mutation alters the assembly of DMVs15 and abolishes viral replication21. Nsp4, 3, and the nuclear (N) protein are located at the DMV16,19, where nsp3 has been shown to form a pore complex that communicates DMV interior with the cytoplasm, that was elusive for some time, pore that could also involve host factors and/or other viral proteins22.\n\nSARS Cov2 recognizes ACE2 (not shown) at the PM of the host cell inducing its endocytosis. Several other entry factors and facilitators have been found to mediate SARS-CoV-2 entry into the host cells. Clathrin-dependent and independent endocytosis mediate viral entry. In the current paradigm, clathrin-mediated endocytosis (middle vesicle at PM) follows the endosomal pathway, that through endosomal acidification and cleavage of S protein by TMPRS2 induces the fusion of SARS-CoV-2 membrane with vesicle membrane deploying vRNA (green stripes) into the cytoplasm. Once released, vRNA reaches cytoplasmic and ER ribosomes starting viral protein synthesis. Viral proteins induce the assembly of RO elements that are interconnected (not shown), initially inducing zER that through bending and scission assemble DMS, CM, and probably DMV, where synthesis of viral proteins and vRNA takes place. Proteins synthesized at ER, DMS and CM reach the Golgi, where they are posttranslationally modified, and the ERGIC, where the viral particles are assembled and set ready for exocytosis (large black arrow, right vesicle at PM). Viral particles have also been observed at multivesicular bodies (not shown). In this paradigm, DMV are believed to derive from DMS and/or CM, although some controversies have been raised (see text), mainly the temporal sequence, the lack of ER markers in DMV and the lack of intermediate structures. VP (not shown) are formed by the fusion of single DMS. In this paradigm, the mitochondrial role is not considered, although evidence has accumulated suggesting its participation (see text).\n\nIn the complementary scenario proposed in this work, DMV are shedded from mitochondria, through a mechanism similar to that described for double-membrane MDV, and or asymmetric mitochondria fission, known to be potentiated after mitochondrial stress. Whether MDV require several transforming steps to become DMV, or if these compartments are essentially the same with viral proteins included needs further investigation (question mark 1). DMV have double-membrane spanning pores (dark blue) in which nsp3 is inserted along with other unidentified molecules. These pores mediate the export of vRNA to the cytoplasm, which complexes with N protein outside DMV. Exported vRNA may be translated immediately by ribosomes located in the external membrane of DMV (question mark 2). Interestingly, some MDV have been shown to carry mitochondrial proteins of the IMM, MOM, and mitochondria matrix, which could be also present at DMV (green dot at MOM, MDV/DMV outer membrane). In this complimentary scenario, a critical question is how vRNA accesses mitochondria (question mark 3). It is possible that vRNA once in the cytoplasm is translated at MOM ribosomes, or that it is translocated into mitochondria through the TOM complex. Alternatively, vRNA could reach mitochondria through the fusion of caveolae with endocytosed SARS-CoV-2, although it is not clear whether coronavirus can follow this pathway. Finally, an intriguing possibility, that could be critical, is whether DMS, which are induced by viral proteins most probably synthesized nearby the ER from where they are derived, may transform into vesicles with viral particles. This possibility is supported by the synthesis of viral proteins at DMS and would require that vRNA is packed inside, which seems feasible because protein synthesis at DMS implicates the presence of vRNA. The lack of de novo synthesized vRNA at DMS indicates that vRNA synthesis does not happen there, but does not rule out the presence of vRNA within DMS. Inset, red square left bottom: A close-up of a DMV with vRNA in its interior (3 green stripes), with one vRNA being exported by the double-membrane spanning pore (dark blue at DMV membranes), complexing with the N protein (light blue). This exported vRNA could be translated in situ at DMV ribosomes (red at DMV external membrane). Peptides synthesized by DMV ribosomes are shown attached in blue and the green dot at DMV represents mitochondrial molecules located at DMV. Abbreviations: CM-convoluted membranes; DMS-double-membrane spherules; DMV-double-membrane vesicles; ER-endoplasmic reticulum; ERGIC-ER-Golgi intermediate compartment; M-mitochondria; PM-plasma membrane; TOM-translocase of the mitochondrial outer membrane; VP-vesicle packet (not shown); vRNA-viral RNA; zER-zippered ER.\n\nMost SARS-CoV-2-induced membrane modifications are derived from ER membranes and are interconnected. In the case of DMV, this origin was in part assumed because such mechanism is presumed to mediate RO assembly in other positive sense RNA viruses23; because they have contacts with other membrane modifications of the RO and the ER, because ribosomes have been observed on DMV surface, and because DMS and CM were proposed to be precursors of DMV11,16,19,20,23. DMV sizes range from 150 to 300 nm, but they grow through infection, and it has been established that SARS-CoV-2 DMV are the location where vRNA synthesis occurs16,19,24. Importantly, DMV are early (1–2 h post-infection [p.i.]) observed in the cell cytoplasm after coronavirus infection in vitro, and their number increase through time reaching a maximum in 6–8 h p.i.16,25. There are currently two models for DMV assembly from the ER. In the case of coronavirus, DMV are thought to be assembled from zippered ER that folds and closes in response to vRNA, as observed with IBV11,23, with CM and DMS as putative intermediate precursors16,19. However, major challenges remain for this model, because no intermediate structures have been recognized between DMV and DMS or CM, and because CM and DMS, both derived from the ER, have no relationship with DMV beyond their membrane contacts16,19. DMV do not have ER (nor ERGIC, Golgi, or endosomal/lysosomal) markers as it would be expected if they were assembled from the ER, and most importantly, it has been shown that DMV assembly (1–2 h p.i.) precedes CM assembly (~3 h p.i.)25. Furthermore, CM and DMS do not carry out vRNA synthesis as DMV do19. Thus, CM and DMS are not DMV precursors19. On the other hand, the high energetic and complex topological requirements assumed to occur for DMV assembly through the zippered ER−CM−DMS model, given their numbers after a few hours post-infection, further complicate this notion. Indeed, it has been suggested that DMV could have another origin than the ER8,11,19.\n\n\nThe mitochondria and double-membrane MDV as the putative origin of SARS-CoV-2 DMV\n\nGiven the recent findings that relate SARS-CoV-2 with mitochondria, it is possible that MDVs are the origin of DMV. MDV were discovered more than a decade ago and they comprise different vesicles shed from mitochondria involved in its quality control14,26. Interestingly, some MDV have double-membrane with a size of 60–150 nm and are shed independently of drp1, mitochondria fission, and autophagy (Figure 2A)26,27. Interestingly, several coronavirus proteins have been shown to down- or up-regulate drp1 (N/envelope [E], nsp3, nsp4a, nsp4b, and ofr9b)10. MDV are generated in steady conditions and have been observed in vivo28, but their number increases after mitochondrial stress or higher respiratory activity26,27. In this regard, it has been found that SARS-CoV-2-infected monocytes have compromised mitochondrial function and energy deficit30,31. This could be the long-term result of viral infection in which mitochondria shedding of MDV/DMV, triggered initially by a burst of metabolic activity or stress, leads to mitochondria function impairment.\n\nA TEM of three isolated mitochondria from bovine heart shedding double membrane vesicles (bar=500 nm in first panel and =100 nm in panels 2 and 3). (Taken ftom 29). B TEM analysis of mitochondria morphology and DMV formation in infectious clone virus (icv)-infected cells (derived from coronavirus murine hepatitis virus; MHV). This icv has a mutated nsp4-N258T that results in temperature sensitive viral replication. As observed, after 5.5 h p.i at the pemissive temperature, DMV are evident (arrows) and some mitochodria (asterisk) are associated with shedded vesicles (left panel, arrowheads and inset). Interestingly, if cells are left at the non-permissive temperature the last 2 h. (right panel), mitochondria appear swollen with enlarged cisternae, and accompanied by increased localization of nsp3 and nsp4 at mitochondria (not shown).(Modified from 15 with permission) (bar=1000 nm). C TEM and autoradiography of de novo synthesized vRNA in SARS CoV infected-Vero E6 cells at 7 h p.i. As observed, some DMV are closely located to mitochondria, showing both vRNA label within. Interestingly, vRNA label in DMV and mitochondria are neighboring (inset) (Modified from 19). D Electron tomography images of an Infectious Bronchitis Virus-infected cell showing a DMV (arrowhead) connected to the ER (arrow) (upper panel). In a different plane (lower panel), the same DMV shows a cisternae-like arrangement of the inner membrane (asterisk), that resembles those of mitochondria, hinting the putative mitochondrial origin of DMV (Modified from 23 with permission). E TEM and autoradiography of de novo synthesized vRNA in SARS-CoV-infected Vero E6 cells at 12 h p.i. It can be observed that DMV (asterisks) are densely labeled with vRNA signal. Notably, a couple of mitochondria are also labeled for vRNA, but importantly, this label is polarized in both mitochondria near the membrane towards the DMV cluster. Intriguilgly, within this pool of DMV, a degenerated mitochondria-like structure is observed (spark at the center), perhaps resulting from extensive DMV shedding (bar=500 nm) (Modified from 19).\n\nMDV not only look like DMV for their double-membrane, they also transport selective cargo to peroxisomes and the endolysosomal system after mitochondrial stress, depending on Vps35 and syntaxin-17/SNAP29/VAMP7, respectively26,27,32,33. This offers a pathway that could be involved in the intracellular transport of viral components to secondary vesicular structures (i.e. ERGIC, lysosomes, multivesicular bodies) where viral particles are assembled and set ready for exocytosis. Interestingly, PINK1/Parkin and the mitochondrial ubiquitin ligase MULAN1 (MAPL) have been involved in MDV shedding from mitochondria27,34. Notably, MULAN1 is known to be involved in the antiviral response of mitochondria35. In addition, some MDV contain mitochondrial molecules, including the translocase complex (specifically TOM 20), components of the OXPHOS complexes, the VDAC, and/or pyruvate dehydrogenase26,29. This opens the possibility that other mitochondrial components and resources for vRNA replication are transported or generated within MDV, such as proteins involved in the translocation of metabolites and solutes since MDV are like “chunks” of mitochondria. In this regard, it was recently shown that DMV have pores that span the double-membrane that mediate the export of vRNA from DMV and could also mediate the exchange of molecules between DMV and the cytoplasm22. These authors also showed that once vRNA is exported, it complexes with viral N protein, association that is known to increase vRNA translation in trans36, which could probably occur at ribosomes located at the external membrane of DMV16. It is important to note that MDV have a very short shedding dynamic (~30 s), and peak at 2 h after stimulation26,34, this could explain why the shedding step is not frequently spotted by TEM. Interestingly, in a recent paper, an alternative mechanism of mitochondria fission has been described that occurs under stress and high energy demand, that depends upon the MOM molecule Fis1, which yields small mitochondria destined for mitophagy37. A common feature of RO are nearby mitochondria, which may show signs of cisterna swelling and disorganization, similar to mitochondria with induced MDV shedding26,27,29,32, or membrane continuity with DMV16,19,23–25. Nevertheless, some TEM images have shown budding of what could be DMV from mitochondria (Figure 2B). In some cases, in closely located DMV and mitochondria, vRNA signal can be observed within both, apposed to each other (Figure 2C). Moreover, in nsp4 temperature-sensitive mutants, at a non-permissive temperature, there is an increase in mitochondria size, with enlarged cisternae, and increased localization of nsp4 and nsp3 at mitochondria, perhaps resulting from the reduction of MDV shedding, that in turn results in a reduced number of DMV15. Interestingly, electron tomography has shown what could be an intermediate between DMV and MDV, a vesicle tethered to the ER with a double-membrane that contains a cisterna-like arrangement of the inner membrane (Figure 2D)23. Further support for the notion that mitochondria could be targets of SARS-CoV-2 vRNA infection that leads to DMV assembly, comes from the observation that shows mitochondria containing newly synthesized vRNA19. This suggests that mitochondria somehow get vRNA that could induce stress and therefore shedding of MDV (/DMV), where the vRNA replication machinery and newly synthesized vRNA are mostly located19. Interestingly, it was recently found that Fe-S cofactors, of which biosynthesis initiates at the mitochondria, are involved in SARS-CoV-2 RdRp function38. Moreover, a recent in silico analysis predicted SARS-CoV-2 RNA localization to host mitochondria and nucleolus, further supporting this idea39. In addition, it is possible that the abundance of vRNA and viral proteins within mitochondria are under tight control through the shedding of MDV, and thus, that only a few vRNA are found within mitochondria at a given moment. Interestingly, some images have shown that vRNA located inside of mitochondria is polarized towards DMV pools (Figure 2C and E)19.\n\nSupporting also the role of mitochondria for DMV assembly is the unexpected identification of several mitochondria molecules involved in different mechanisms of its physiology (electron transport, metabolism, mitochondria ribosomes, RNA maturation, and cellular immune signaling) as interactors of viral proteins40. Some of the putative relevant interactions that this work identified is that of nsp4 with the inner mitochondria membrane translocase (TIMM) complex, the interaction of ORF9b with TOMM70, and the interaction of nsp6 and ORF9c with the Sigma receptor. This receptor has been involved in several mitochondria functions, related to its location at the mitochondria-associated ER membranes (MAM)41, enriched with interactors of nsp2 and 4. Additional intriguing, unexpected interactions were those of SARS CoV-2 membrane (M) protein with FASTKD5, involved in mitochondrial RNA maturation, and that of nsp8 with different mitochondria ribosomal proteins (MRP). Strikingly, interactions of ORF3a and M protein with relatives of known partners of MULAN1 (REEP and TRIM) were also identified. In a different study, nsp2 was found to interact with VDAC242, the mitochondrial porine, whereas the mitochondria antiviral-signaling protein (MAVS) has also been identified as a target of SARS-CoV-2 infection10. Together, these interactions of viral proteins with the host support that mitochondrial function is very relevant for SARS-CoV-2 infection. Furthermore, since the ribosome, mitochondrial RNA maturation and translocation mechanisms are targets of viral proteins, that according to the current model of infection are unexpected, these findings also hint that SARS-CoV-2 infects mitochondria as part of its replication cycle, rather than only taking control of this organelle through viral proteins synthesized elsewhere. The down-regulation of mtDNA encoded genes and mitochondrial RNA in patient autopsies by SARS-CoV-2 also supports this notion43. Other relevant interactions of the viral proteome with mitochondrial proteins have been analyzed by others8,10,42. A key question in this scenario is which are the steps that mediate the shedding and transformation of MDV into DMV, and how viral proteins are involved (Figure 1 question mark 1).\n\n\nMitochondria infection by vRNA\n\nHow MDV–DMV are induced by SARS-CoV-2? This question has no answer yet; however, there are at least two main possibilities that are non-self-exclusive: one that is consistent with the current paradigm is that viral proteins synthesized at the ER and/or its membrane modifications in the RO somehow reach mitochondria, modulate its physiology and induce DMV, in 1–2 hours. In this regard, there are some viral proteins with mitochondria localization sequences such as 3b44, or that target proteins at the inner mitochondria membrane (IMM) (i.e., TIMM, electron transport proteins, and MRPS), at the MOM (TOM), or at the mitochondria matrix (FASTKD5). Alternatively, the mitochondria could start viral protein synthesis with ribosomes located at the MOM45, and/or uptake vRNA from the cytoplasm after virus entry and vRNA release into the cytoplasm. In this regard, it is known that mitochondria are capable of importing RNA from the cytoplasm through a pathway that involves the TOM/TIMM complex46, and SARS-CoV-2 RNA is predicted to locate at this organelle39. On the other hand, a tantalizing possibility is that vRNA accesses mitochondria directly from vesicles shed from the plasma membrane (PM) in which SARS-CoV-2 is endocytosed. This PM–mitochondria pathway mediates caveolin transport to mitochondria in myocytes after stress47 (Figure 3A), and it could be an early step of what we called plasma membrane-mitochondria bridges, which we recently described in astrocytes (Figure 3B)48, involved in the emerging pathway of PM–mitochondria interactions49. These PM-mitochondria bridges contain vesicles, most probably caveolae and mediate mass transfer from PM to mitochondria in minutes. Given that DMV are induced early by coronavirus (1–2 h p.i.), direct access of vRNA to mitochondria seems plausible, providing also the possibility to synthesize some viral proteins at IMM tethered mitoribosomes, with which nsp8 interacts40. Notably, mitoribosomes synthesize most exclusively membrane proteins that are co-translationally inserted into the membrane with the participation of OXA150,51, as it is the case of transmembrane-containing nsp3, 4 and 6, involved in vRNA replication, of which nsp3 and 4 have been located at DMV and colocalize with nsp2, 5, 8, 12, 13 and 1516,19,22,24,25. Interestingly, the M protein could optimize vRNA translation through its interaction with mitochondrial FASTKD5 protein40, involved in mitochondrial RNA maturation50, and at the same time, viral replication could profit the mitochondria synthesized Fe-S cofactors required for RdRp function38. This scenario could provide the advantage of the protected environment of mitochondria matrix, rich in ATP, avoiding the requirement of large amounts of protein to be transported from ER to mitochondria that would require energy and time.\n\nA TEM image of a cardiac myocyte in which caveolae are closely apposed to mitochondria. In this work it was found that PM–mitochondria transfer of caveolin increased cellular fitness against ischemia-reperfusion (scale not-defined) (Taken with permission from 45). B TEM of PM–mitochondria bridges that we recently described in cultured astrocytes. These structures consist of a highly electrodense region between PM and mitochondria (arrowheads) which is associated with vesicles with the size of caveolae (arrow), flattening of the mitochondria membrane facing the PM, and dots within mitochondria that also presents cisternae perpendicular to the PM, similar to mitochondria adherens complex (MAC) in neurons56. These structures mediated the mass transfer from PM to mitochondria in minutes (bar=250 nm) (Taken with permission from 46).\n\nHowever, the main concern against the idea that mitochondria can directly uptake SARS-CoV-2 from PM caveola comes from one study suggesting that SARS-CoV endocytosis is caveolin-independent52. This finding is based on the observation that cholesterol sequestration (one of the main components of lipid rafts that in turn is endocytosed by caveolae,50) with nystatin and filipin did not block pseudovirus entry. Indeed, nystatin enhanced it, whereas another cholesterol sequestering molecule, MβCD, did block it, therefore raising doubts about how cholesterol is involved. As matter of fact, different mechanisms of endocytosis have been found to mediate SARS-CoV-2 internalization52,53–55. In addition, in this study and a different one with CoV NL63, a major lack of colocalization between viral spike protein and caveolin-1 at 20 or 60 min p.i. was also considered as evidence for a caveolin-independent mechanism. However, the putative fast dynamic nature of this interaction (since we found that mass is transferred from PM to mitochondria in ~2 min) nor extracellular conditions were considered in these approaches. Extracellular conditions are expected to be acidified in the inflammatory setting and the extracellular acidification rate (ECAR) has been found increased in SARS-CoV-2 infected monocytes31. Importantly, extracellular acidification is known to induce transfer of caveolae to mitochondria (Reviewed in 47). In addition, some evidence supports a role of the caveolae pathway in SARS-CoV-2 endocytosis: a) lipid rafts integrity is required for SARS-CoV entry and ACE2 is localized into lipid rafts, that are endocytosed through caveolae, well-known signaling hubs54,57; b) the S protein co-fractionates with caveolin-1 after binding to ACE254; c) an in silico approach found that SARS-CoV-2 proteins S, M, orf3, and replicase 1AB have putative caveolin binding motifs58; and d) orf3a protein binding to caveolin has been demonstrated experimentally59. Thus, the precise role of cholesterol, caveolae, and caveolin for SARS-CoV-2 infection requires further investigation, because direct viral targeting to mitochondria could be of great relevance for SARS-CoV-2 infection. Interestingly, cholesterol-bound RNA probes are targeted to mitochondria60. Furthermore, several alternative entry factors to ACE2 and facilitators capable to mediate SARS-CoV-2 infection have been identified61–64, and they could mediate SARS-CoV-2 caveolae-mediated endocytosis. Taken together, it is possible that the diversity of receptors and entry pathways exploited by SARS-CoV-2, together with the fast dynamics of PM–mitochondria communication can obscure the caveolae role.\n\n\nConclusions\n\nAccording to the published literature, it seems possible to conceive that SARS-CoV-2 DMV have a mitochondrial origin, through the shedding of MDV as shown in Figure 1. This possibility is supported by different observations reviewed here and would include mitochondria infection by vRNA, which could occur by its uptake from the cytoplasm through the TOM complex or direct targeting to mitochondria of PM caveolae containing SARS-CoV-2 (Figure 1, question mark 3), as it has been reported in myocytes and astrocytes. A major advantage of the proposed role of mitochondria in DMV assembly, in comparison with their origin from the ER, is the shortest time to induce DMV, since protein synthesis required to induce ER zippering and bending would not be necessary until later when the ER is infected by vRNA. However, still many questions remain in this scenario, and most probably, previous findings that escaped this review may challenge this hypothesis, that nevertheless pretends to be an integrative starting point to further examine SARS-CoV-2 infectious cycle. For instance, is it possible that contacts between DMV and other RO-modified membranes could be related to MAM?, structures that mediate ER–mitochondria interactions and are critical for their function41. It is important also to elucidate the steps that promote MDV shedding after vRNA infection and how these MDV transform into DMV (Figure 1, question mark 1). In this regard, it is also possible that other mitochondrial molecules can be present at MDV/DMV, that increase viral fitness. Another relevant question is the origin of ribosomes that decorate DMV, that could assemble de novo with the action of viral proteins similar to MOM-tethered ribosomes45, that are related with the PINK1/Parkin pathway, and whether they are involved in the immediate translation of vRNA after its export from DMV (Figure 1, question mark 2). Also, the identification of FASTKD5 as an interactor of M protein opens the possibility that within mitochondria, vRNA could be target of maturation, which in turn could optimize viral protein synthesis at mitoribosomes, or when this processed vRNA are exported from DMV. In this regard, codon variation in the human mitochondria genetic code could provide clues that support or reject this hypothesis65. Another intriguing possibility that should be tested is whether DMS eventually become vesicles with virions inside (Figure 1, question mark 4). This is because, the fate of DMS is not clear, however, given the topology of their membranes and the nearby synthesis of viral proteins (perhaps in the interior of DMS), it is conceivable that the closed inner membrane becomes the viral membrane, deployed later to the ERGIC. Also pending is whether CM are the byproduct of DMV or DMS, as it has been proposed19,25. CM could be collapsed DMV that exhausted available resources in their vicinity, since CM increase after DMV formation slows down25, and/or debris that remains after DMS assembly. Both mechanisms are consistent with the accumulation of viral proteins at CM. In addition, could this alternative pathway of SARS-CoV-2 cell infection be related with the lack of effect of drugs that target the endolysosomal pathway? All these questions require further research to be answered and would test this complimentary model of SARS-CoV-2 infection of mitochondria and DMV assembly. Nevertheless, it seems clear that a diversity of cellular mechanisms (entry factors and facilitators, endocytosis, cleaving proteases, organelles) are exploited by SARS-CoV-2 to infect cells, replicate and succeed.\n\nGiven the pandemic emergency worldwide, a deeper understanding of the cellular mechanisms that are exploited by SARS-CoV-2 to infect cells seems urgent as it could lead to envisage novel therapeutic targets and alternatives to control or stop the pandemic that today is still enhancing the death toll. The model proposed here for SARS-CoV-2 infection and DMV assembly provides a non-conventional scenario to explore, that could help to treat or prevent SARS-CoV-2 infection, for instance with mitochondria-targeted molecules (i.e. chloramphenicol alone or in combination with other drugs; mitochondria-targeted RNA; mitochondria protein/cofactor synthesis and function), some of which have been identified as candidates to treat COVID-1940.\n\n\nAbbreviations\n\nCM-convoluted membranes; DMS-double membrane spherules; DMV-double membrane vesicels; ER-endoplasmic reticulum; ERGIC-ER-Golgi intermediate compartment; IMM-inner mitochondria membrane; M-mitochondria; MAM-mitochondria associated membrane; MAVS-mitochondria antiviral-signaling protein; MOM-mitochondria outer membrane; nsp-non-structural protein; nsp-non-structural protein; PM-plasma membrane; TIMM-translocase of the mitochondrial inner membrane; TOM-translocase of the mitochondrial outer membrane; VDAC-Voltage-dependent anion channel; VP-vesicle packet (not shown); vRNA-viral RNA; zER-zippered ER.\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\nAuthors' contributions\n\nPMOB did all work related with this manuscript.", "appendix": "Acknowledgements\n\nPMOB wishes to thank Ph. D. Diana Elinos Calderon for her instigating questions regarding this hypothesis, as well as family members for available time shortages and my science insistence despite adverse conditions at INNN and Mexico.\n\n\nReferences\n\nWHO: WHO Coronavirus Disease (COVID-19) Dashboard. 2021. Reference Source\n\nBerlin DA, Gulik RM, Martinez FJ: Severe Covid-19. N Engl J Med. 2020; 383(25): 2451–2460. PubMed Abstract \| Publisher Full Text\n\nPAHO: COVID-19 and comorbidities in the Americas: Hands-on tool to estimate the population at increased and high risk of severe COVID-19 due to underlying health conditions for the Americas. iris. 2020. Reference Source\n\nHussain A, Kaler J, Tabrez E, et al.: Novel COVID-19: A Comprehensive Review of Transmission, Manifestation, and Pathogenesis. Cureus. 2020; 12(5): e8184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiersinga WJ, Rhodes A, Cheng AC, et al.: Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review. JAMA. 2020; 324(8): 782–793. PubMed Abstract \| Publisher Full Text\n\nParasher A: COVID-19: Current understanding of its pathophysiology, clinical presentation and treatment. Postgrad Med J. 2021; 97(1147): 312–320. PubMed Abstract \| Publisher Full Text\n\nOronsky B, Larson C, Hammond TC, et al.: A Review of Persistent Post‑COVID Syndrome (PPCS). Clin Rev Allergy Immunol. 2021; 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh KK, Chaubey G, Chen JY, et al.: Decoding SARS-CoV-2 hijacking of host mitochondria in COVID-19 pathogenesis. Am J Physiol Cell Physiol. 2020; 319(2): C258–C267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurtscher J, Cappellano G, Omori A, et al.: Mitochondria: In the Cross Fire of SARS-CoV-2 and Immunity. iScience. 2020; 23(10): 101631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGatti P, Ilamathi HS, Todkar K, et al.: Mitochondria Targeted Viral Replication and Survival Strategies-Prospective on SARS-CoV-2. Front Pharmacol. 2020; 11: 578599. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolff G, Melia CE, Snijder EJ, et al.: Double-Membrane Vesicles as Platforms for Viral Replication. Trends Microbiol. 2020; 28(12): 1022–1033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanchard E, Roingeard P: Virus-induced double-membrane vesicles. Cell Microbiol. 2015; 17(1): 45–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaul D, Bartenschlager R: Architecture and Biogenesis of plus-strand RNA virus Replication Factories. World J Virol. 2013; 2(2): 32–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSugiura A, McLelland GL, Fon EA, et al.: A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 2014; 33(19): 2142–2156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClementz MA, Kanjanahaluethai A, O'Brien TE, et al.: Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles. Virology. 2008; 375(1): 118–129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnoops K, Kikkert M, van der Worm SH, et al.: SARS-Coronavirus Replication is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum. PLoS Biol. 2008; 6(9): e226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKopek BG, Perkins G, Miller DJ, et al.: Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a virus-induced mini-organelle. PLoS Biol. 2007; 5(9): e220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomasundaran M, Zapp ML, Beattie LK, et al.: Localization of HIV RNA in Mitochondria of Infected Cells: Potential Role in Cytopathogenicity. J Cell Biol. 1994; 126(6): 1353–1360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnijder EJ, Limpens RWAL, de Wilde AH, et al.: A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis. PLoS Biol. 2020; 18(6): e3000715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngelini MM, Akhlaghpour M, Neuman BW, et al.: Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles. mBio. 2013; 4(4): e00524–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSakai Y, Kawachi K, Terada Y, et al.: Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication. Virology. 2017; 510: 165–174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolff G, Limpens RWAL, Zevenhoven-dobbe JC, et al.: A molecular pore spans the double membrane of the coronavirus replication organelle. Science. 2020; 369(6509): 1395–1398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaier HJ, Hawes PC, Cottam EM, et al.: Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes. mBio. 2013; 4(5): e00801–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnijder EJ, van der Meer Y, Zevenhoven-Dobbe J, et al.: Ultrastructure and Origin of Membrane Vesicles Associated with the Severe Acute Respiratory Syndrome Coronavirus Replication Complex. J Virol. 2006; 80(12): 5927–5940. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUlasli M, Verheije MH, de Haan CA, et al.: Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus. Cell Microbiol. 2010; 12(6): 844–861. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeuspiel M, Schauss AC, Braschi E, et al.: Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers. Curr Biol. 2008; 18(2): 102–108. PubMed Abstract \| Publisher Full Text\n\nSoubannier V, McLelland GL, Zunino R, et al.: A vesicular transport pathway shuttles cargo from mitochondria to lysosomes. Curr Biol. 2012; 22(2): 135–141. PubMed Abstract \| Publisher Full Text\n\nCadete VJ, Deschênes S, Cuillerier A, et al.: Formation of mitochondrial-derived vesicles is an active and physiologically relevant mitochondrial quality control process in the cardiac system. J Physiol. 2016; 594(18): 5343–5362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoubannier V, Rippstein P, Kaufman BA, et al.: Reconstitution of Mitochondria Derived Vesicle Formation Demonstrates Selective Enrichment of Oxidized Cargo. PLoS One. 2012; 7(12): e52830. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAjaz S, McPhail MJ, Singh KK, et al.: Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral blood mononuclear cells of patients with COVID-19. Am J Physiol Cell Physiol. 2021; 320(1): C57–C65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibellini L, De Biasi S, Paolini A, et al.: Altered bioenergetics and mitochondrial dysfunction of monocytes in patients with COVID-19 pneumonia. EMBO Mol Med. 2020; 12(12): e13001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLelland GL, Lee SA, McBride HM, et al.: Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system. J Cell Biol. 2016; 214(3): 275–291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraschi E, Goyon V, Zunino R, et al.: Vps35 Mediates Vesicle Transport between the Mitochondria and Peroxisomes. Curr Biol. 2010; 20(14): 1310–1315. PubMed Abstract \| Publisher Full Text\n\nMcLelland GL, Soubannier V, Chen CX, et al.: Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. EMBO J. 2014; 33(4): 282–295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJenkins K, Khoo JJ, Sadler A, et al.: Mitochondrially localised MUL1 is a novel modulator of antiviral signaling. Immunol Cell Biol. 2013; 91(4): 321–330. PubMed Abstract \| Publisher Full Text\n\nNakagawa K, Lokugamage KG, Makino S: Viral and Cellular mRNA Translation in Coronavirus-Infected Cells. Adv Virus Res. 2016; 96: 165–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKleele T, Rey T, Winter J, et al.: Distinct fission signatures predict mitochondrial degradation or biogenesis. Nature. 2021; 593(7859): 435–439. PubMed Abstract \| Publisher Full Text\n\nMaio N, Lafont BAP, Sil D, et al.: Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets. Science. 2021; 373(6551): 236–241. PubMed Abstract \| Publisher Full Text\n\nWu KE, Fazal FM, Parker KR, et al.: RNA-GPS Predicts SARS-CoV-2 RNA Residency to Host Mitochondria and Nucleolus. Cell Syst. 2020; 11(1): 102–108.e3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGordon DE, Jang GM, Bouhaddou M, et al.: A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature. 2020; 583(7816): 459–468. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee S, Min KT: The Interface Between ER and Mitochondria: Molecular Compositions and Functions. Mol Cells. 2018; 41(12): 1000–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavies JP, Almasy KM, McDonald EF, et al.: Comparative Multiplexed Interactomics of SARS-CoV-2 and Homologous Coronavirus Nonstructural Proteins Identifies Unique and Shared Host-Cell Dependencies. ACS Infect Dis. 2020; 6(12): 3174–3189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller B, Silverstein A, Flores M, et al.: Host mitochondrial transcriptome response to SARS-CoV-2 in multiple cell models and clinical samples. Sci Rep. 2021; 11(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuan X, Shan Y, Yao Z, et al.: Mitochondrial Location of Severe Acute Respiratory Syndrome Coronavirus 3b Protein. Mol Cells. 2006; 21(2): 186–191. PubMed Abstract\n\nLu B, Guo S: Mechanisms Linking Mitochondrial Dysfunction and Proteostasis Failure. Trends Cell Biol. 2020; 30(4): 317–328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamenski PA, Krasheninnikov IA, Tarassov I: 40 Years of Studying RNA Import into Mitochondria: From Basic Mechanisms to Gene Therapy Strategies. Mol Biol (Mosk). 2019; 53(6): 924–932. PubMed Abstract \| Publisher Full Text\n\nFridolfsson HN, Kawaraguchi Y, Ali SS, et al.: Mitochondria-localized caveolin in adaptation to cellular stress and injury. FASEB J. 2012; 26(11): 4637–4649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMontes de Oca Balderas P, Matus Nuñez M, Picones A, et al.: NMDAR in cultured astrocytes: Flux-independent pH sensor and flux-dependent regulator of mitochondria and plasma membrane-mitochondria bridging. FASEB J. 2020; 34(12): 16622–16644. PubMed Abstract \| Publisher Full Text\n\nMontes de Oca-B P: Mitochondria Plasma Membrane Interactions. JBC (in Press). 2021.\n\nKummer E, Ban N: Mechanisms and regulation of protein synthesis in mitochondria. Nat Rev Mol Cell Biol. 2021; 22(5): 307–325. PubMed Abstract \| Publisher Full Text\n\nItoh Y, Andréll J, Choi A, et al.: Mechanism of membrane-tethered mitochondrial protein synthesis. Science. 2021; 371(6531): 846–849. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang H, Yang P, Liu K, et al.: SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res. 2008; 18(2): 290–301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilewska A, Nowak P, Owczarek K, et al.: Entry of Human Coronavirus NL63 into the Cell. J Virol. 2018; 92(3): e01933–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu Y, Liu DX, Tam JP: Lipid rafts are involved in SARS-CoV entry into Vero E6 cells. Biochem Biophys Res Commun. 2008; 369(2): 344–349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInoue Y, Tanaka N, Tanaka Y, et al.: Clathrin-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus into Target Cells Expressing ACE2 with the Cytoplasmic Tail Deleted. J Virol. 2007; 81(16): 8722–8729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins GA, Tjong J, Brown JM, et al.: The Micro-Architecture of Mitochondria at Active Zones: Electron Tomography Reveals Novel Anchoring Scaffolds and Cristae Structured for High-Rate Metabolism. J Neurosci. 2010; 30(3): 1015–1026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng JBX, Nichols BJ: Caveolae: One Function or Many? Trends Cell Biol. 2016; 26(3): 177–189. PubMed Abstract \| Publisher Full Text\n\nCai QC, Jiang QW, Zhao GM, et al.: Putative caveolin-binding sites in SARS-CoV proteins. Acta Pharmacol Sin. 2003; 24(10): 1051–1059. PubMed Abstract\n\nPadhan K, Tanwar C, Hussain A, et al.: Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J Gen Virol. 2007; 88(Pt 11): 3067–3077. PubMed Abstract \| Publisher Full Text\n\nDovydenko I, Tarassov I, Venyaminova A, et al.: Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds. Biomaterials. 2016; 76: 408–417. PubMed Abstract \| Publisher Full Text\n\nIchimura T, Mori Y, Aschauer P, et al.: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney. medRxiv. 2020; 2020.09.16.20190694. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang K, Chen W, Zhang Z, et al.: CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells. Signal Transduct Target Ther. 2020; 5(1): 283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarabipour S, Mac Gabhann F: Targeting neuropilins as a viable SARS-CoV-2 treatment. FEBS J. 2021; 288(17): 5122–5129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLempp FA, Soriaga L, Montiel-Ruiz M, et al.: Lectins enhance SARS-CoV-2 infection and influence neutralizing antibodies. Nature. 2021. PubMed Abstract \| Publisher Full Text\n\nGray MW, Burger G, Lang BF: Mitochondrial Evolution. Science. 1999; 283(5407): 1476–1481. PubMed Abstract \| Publisher Full Text" }	[ { "id": "124209", "date": "16 Mar 2022", "name": "Marc Germain", "expertise": [ "Reviewer Expertise Mitochondria", "MDV", "endoplasmic reticulum", "lysosomes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 argues that the double-membrane vesicles (DMVs) required for coronavirus replication could originate from mitochondria-derived vesicles (MDVs). This is an intriguing possibility that, if true, would open new research avenues. However, there are a number of holes in the argumentation that required to solidify the hypothesis.\nDVMs are generally assumed to originate from the ER. The author argues that because of the shortcomings of this hypothesis, we should be looking for a different source for these membranes. However, the proposed MDV origin also comes with its own issues and unknown. The strengths and weaknesses of each model should be directly compared to allow the reader to properly assess both.\n\nSimilarly, the potential role of ER-mitochondria contact sites in viral RNA replication should be better discussed.\n\nThe description of MDVs does not really reflect the complexity of these vesicles. Several types of MDVs exist, each with its own cargo and destination, and not all of them contain oxidized mitochondrial content destined for degradation in response to mitochondrial stress. What kind of MDVs are envisioned as the source of DMVs (TOM20-positive outer membrane vesicles? Vesicles with oxidized cargo? MAPL-positive MDVs? Other types of MDVs?). Also, MDVs are DRP1-dependent (doi: 10.1038/s41556-021-00798-4).\n\nThe author makes a compelling point for the involvement of mitochondria in coronavirus infection, but this does not necessarily extend to MDVs for which the evidence is much weaker.\n\nIt should be made clear in the text that the interactions reported in ref 40 are for the most part putative as they have not been experimentally confirmed. Also, some of the potential interaction need to be more carefully described. For example, TRIM and REEP are large family of proteins with various members having distinct functions and interacting partners.\n\nSome references are missing (transfer from PM to mitochondria in minutes, p.7) or wrong (figure 3B is from ref 48, not 46; ref 45 does not discuss viral proteins).\n\nHow viral RNA or protein synthesis would be targeted to mitochondria is unclear. Ribosomes are recruited to mitochondria because the proteins they are translating contain a mitochondrial-targeting sequence (MTS) that bind to the TOM complex. Are there MTS on the viral proteins targeted to mitochondria?\n\nWhat would be the membrane topology resulting from the caveolin-dependent delivery of viral particles to mitochondria? How would nsp3/4/6 reach the inner membrane? In this context, where would the precursor polyprotein be cleaved to generate the individual proteins?\n\nMinor point: in the introduction, it is rather strange to state that different vaccine strategies are under development when, in fact, vaccines have been used for over a year.\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? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "8000", "date": "01 Apr 2022", "name": "Pavel Montes de Oca-B", "role": "Author Response", "response": "First, I want to deeply thank Dr. Marc Germain for reviewing the manuscript. Below, each point raised is tackled and appropriate modifications and additions will be included in the following version of the manuscript, that will also include the issues raised by a second reviewer. Beforehand, I apologize if these modifications are not immediately performed in the manuscript, since considering all comments and questions from reviewers as a whole will be much easier, practical, and integrative for a new version of the manuscript. The main concerns with the ER origin of DMV are: the topological needs that such origin implicates and the high energetic cost that such model would require. That is, if one considers the ER origin according to the proposed model described by Mihlec et al., 2021, it seems clear that the movement of ER membranes required to first form and then close DMV seem to be rather complex, mainly at the closing point, because it is required that a 2 dimensional plane is folded to converge into a single closing point, to then scission the vesicle from the ER, resulting in a sphere (in the case of a single DMV). In this regard, it seems more plausible the mitochondrial origin, because it only requires an evagination and a scission from an organelle that already has a double membrane (that in terms of topology, pose a huge problem with the ER origin). The scission of vesicles continuously occurs in mitochondria under basal conditions and is increased under stressful conditions. Interestingly, such stress may be elicited by mitochondria infection with vRNA, that has been recently demonstrated (Shang et al., 2022). Theis mechanism is less contentious than a complicate series of movements and foldings from a 2-dimensional plane, that most probably would require the involvement of the cytoskeleton and molecular motors. On the other hand, the involvement of molecular motors, required to move membranes, implicates the use of energy, and considering the complicate foldings and movements required in the ER model, as well as the large number of DMV that are induced after infection, then the required energy would be also considerable and could be a limiting step (Interestingly, the SARS CoV-2 infection induces glycolysis and importantly reduces OXPHOS). This complexity contrasts with the rather simple and common evagination and scission from the mitochondria, mechanism of vesicle assembly that occurs all the time, not only at mitochondria level, but also at the plasma membrane and other intracellular organelles, the classical mechanism of vesicle formation. In this regard, the ER origin of DMV would implicate that a completely different mechanism of vesicle formation, not observed in cells to my knowledge, is induced by the virus. Despite this is not impossible, from the evolutionary point of view, it seems improbable that a unique, complex and costly mechanism has been selected along with the efficient infective coronavirus, over the common cellular mechanism of vesicle formation. Finally, it is intriguing that the ER origin of DMV is advocated almost unquestionably when it has been reported the absence of conventional ER markers in DMV, such as protein disulfide isomerase (PDI) or calreticulin (Snijder, et al. 2006; Ulasli et al, 2010; Oostra, et al 2007). In contrast other non-conventional markers such as calnexin or RTN3 have been observed (Hackstadt et al., 2021; Cortese et al. 2021). Nevertheless, and interestingly, these molecules are part of the proteome of mitochondria-ER contacts (Cho et al., 2017). These considerations will be included in the reviewed version of the manuscript. Possibly, these contacts are involved in favoring immediate protein synthesis after vRNA exit from the DMV. Nevertheless, they could also provide Ca2+ to the DMV for RNA synthesis, or lipids to enable DMV growth. Also, it is known that mitochondria-ER contact disruption impairs organelle recycling, that FIS-1 recruits DRP for mitochondria fission, and that they are involved in apoptosis, mitophagy and autophagy induction (Lee and Min, 2018). The regulation of these functions could also be relevant for SARS CoV-2 replication cycle. This requires further research to be solved. These considerations will be included in the reviewed version of the manuscript. Necessarily, in my opinion, it would be double membrane vesicles, a key feature of DMV, and it is feasible that they are those containing the TOM machinery to translocate specific proteins into de DMV/MDV. Whether they may content oxidized cargo needs to be evaluated, because the infection reduces OXPHOS then, perhaps not so many oxidized molecules are generated. The mechanism of induction could be other than oxidation within mitochondria, possibly vRNA infection of mitochondria itself, that has been recently demonstrated for SARS CoV-2 (Shang et al., 2022). The dynamics of OXPHOS decrease and glycolysis induction may shed some light to answer this question. The presence of MAPL/MULAN could occur because it is an interactor of the viral proteome, that nevertheless seems to be non-abundant in MDV. Regarding the role of DRP it seems indeed contra intuitive, since DRP has been reported to be degraded through the action of the viral protein ORF9b (Shi et al., 2014). In this regard it is possible that ORF9b is synthesized later after DMV generation, that occurs early after infection. That is, mitochondria infection induces DMV early, and later after protein synthesis, from de novo synthesized vRNA in DMV, ORF9b induces DRP degradation. Importantly, since the experiments that demonstrated DRP degradation by ORF9b were performed in a cell line that constitutively expresses ORF9b, it is conceivable that this model differs from ORF9b expression in viral infected cells. Furthermore, despite this was an ORF9b overexpression system, DRP was not fully degraded, 30% of total DRP according to authors was not degraded. Thus, the dynamics of DRP degradation induced by ORF9b requires to be confirmed in real-viral infected cells, because even proteasome degradation could be impaired given the high expression of viral proteins. These considerations will be included in the reviewed version of the manuscript. I do agree, since the idea that MDV could originate DMV has not been demonstrated and this manuscript only collects the evidence supporting this idea, more research is required to test the possible involvement of the MDV pathway. Also, it is required to test whether MDV differ from DMV at the molecular level and in their genesis, if it is the case that they are derived from mitochondria. The fact that it has recently been demonstrated that mitochondria is infected by SARS CoV-2 vRNA advocates for MDV as DMV precursors (Shang et al., 2022). Ok, this will be stated more clearly in the following version of the manuscript. Also, the specific interaction of TRIM and REEP with viral molecules will be more detailed and analyzed. Yes, sorry for the mistake. This has been corrected and all references checked. According to this idea, vRNA could be translated full or in part (at least those proteins required for RdRp assembly) at mitochondrial ribosomes within the mitochondria matrix, that are closer to prokaryotic ribosomes. Nevertheless, surface MOM ribosomes could translate de novo vRNA emerging from the DMV. As far as I am aware, no MTS has been reported in viral proteins, although I am not sure that they have been looked for. Nevertheless, for proteins that are translated within the mitochondria no MTS would be necessary. These considerations will be clarified in the reviewed version of the manuscript. The delivery of complete viral particles into mitochondria seems improbable, as it would require the escape of the full viral particle from the vesicle, as well as from the fusion mediated by Spike that enables vRNA translocation to the vesicle external compartment. One odd topological possibility exists nevertheless, the fusion of caveola to the MOM, that would enable the virus fuse with the IMM. However, this is only a topological possibility that seems difficult given the protease and pH needs required to activate Spike protein fusion machinery that may not be fulfilled at the intermembrane space. Rather, I would favor the idea that the vRNA could be translocated into mitochondria, either directly from the caveolae, or after an initial translocation to the cytoplasm. Importantly, vRNA has been demonstrated this year within mitochondria matrix (Shang et al., 2022). These scenarios require further investigation, since plasma membrane-mitochondria interactions have been somewhat disregarded (Montes de Oca B, 2021). You are right, I wrote the initial draft before April 2021, they are still in clinical trials III and IV. This will be corrected in the manuscript." }, { "c_id": "9048", "date": "04 Jan 2023", "name": "Marc Germain", "role": "Reviewer Response", "response": "Thank you for the clarifications. Do you think that the synthesis of viral proteins on mitochondrial ribosomes would be affected by the fact that the mitochondrial genetic code is slightly different from the standard code?" } ] } ]	1	https://f1000research.com/articles/10-1009
https://f1000research.com/articles/13-787/v1	10 Jul 24	{ "type": "Systematic Review", "title": "Physical activity and sedentary behavior perceptions in overweight and obese adults: A systematic review of qualitative study", "authors": [ "Neha Bora", "Vaishali K", "Ashwani Verma", "Avinash Kumar Bharti", "Mukesh Kumar Sinha", "Neha Bora", "Vaishali K", "Ashwani Verma", "Avinash Kumar Bharti" ], "abstract": "Background One of the largest hazards to human health is obesity, which is intimately related to sedentarism and physical inactivity. Understanding the perspectives and attitudes of adults with overweight or obesity towards their lifestyle choices related to sedentary behavior and physical activity is essential for mitigating associated health risks.\n\nObjective This systematic review aims to collate the extent of qualitative research on the perception of sedentary behavior and physical activity among adults with overweight and obesity.\n\nMethods A comprehensive search of Scopus, PubMed, CINAHL, and Web of Science, databases was conducted, which yielded 2,881 articles. A total of 2591 abstracts were screened, and 45 full-text articles were examined. A total of nine qualitative studies involving adults with overweight or obesity (BMI > 25 kg/m2) were included in this systematic review. Data extraction utilized Rayyan.qcri.org software, and studies were critically appraised using Joanna Briggs’s Institute checklist for qualitative research.\n\nResults The included studies revealed a diverse array of themes, wherein a few perceived factors reported towards sedentary behavior were lack of awareness about the hazards, mode of relaxation, family commitment, technology use, motivation deficits, and fatigue. Barriers to physical activity encompassed social, cultural, and environmental factors. In contrast, peer support, fitness facility access, accountability, mental health awareness, well-being, and weight management facilitate physical activity involvement.\n\nConclusion Perceptions in overweight and obese adults on sedentary living and exercise are intricate and multifaceted. This review provides valuable insights that can inform clinicians and researchers in promoting regular physical activity for adults with overweight and obesity.", "keywords": [ "exercise", "sedentary lifestyle", "weight gain", "beliefs", "scoping review" ], "content": "Introduction\n\nObesity, characterized by chronic adipose tissue inflammation and overeating, leads to sustained fat storage and influences various physiological processes.1 It poses significant risks to human health, playing a major role in the surge of non-communicable diseases.2 As per WHO, one out of every eight individual is obese globally in 2022.3 Notably, between 1980 and 2019, the prevalence of obesity experienced a dramatic increase, surging from 3.2% to 12.2% for men and from 6% to 15.7% for women.4 The Global Burden of Disease in 2015 reported the prevalence of obesity had doubled in more than 70 countries, resulted in 4 million deaths from 1980-2015.5 If these trends persist, it is projected that by 2025, 2.7 billion adults will be overweight, and over one billion will be obese globally.4\n\nIn addition to the abundance of endocrine, metabolic, and environmental factors, it is presently understood that the rise of the global obesity epidemic in recent decades is primarily attributed to behavioral factors such as sedentary living and a decrease in overall physical activity (PA), coupled with the consumption of unhealthy diets.6 Individuals who are overweight and obese often exhibit higher levels of sedentary behavior (SB),7 characterized by activities involving minimal energy expenditure (around 1.5 Metabolic Equivalent Units) while sitting or lying down.8 This SB not only contributes to additional weight gain but also leads to reduced PA levels, perpetuating the cycle of obesity.8\n\nAddressing obesity’s impacts can be significantly alleviated through lifestyle modifications, including increased PA, adopting a nutritious diet, ensuring sufficient sleep, and reducing SB.2 The World Health Organization promotes healthy eating and regular exercise as effective and accessible means to prevent and manage overweight and obesity and to mitigate the burden of non-communicable diseases.2 Despite knowing the importance and health benefits of PA, individuals with overweight and obesity are shown to be less physically active than adults with normal weight.9 Thus, to address sufficient PA in this population, it is important to understand the perspective of individuals who are overweight or obese regarding their lifestyle. Systematic reviews are conducted on factors facilitating and limiting PA in various resource settings and low-middle-income countries. However, little is known about the perceptions of PA and SB at the global level from different countries and the differences noted in these countries. This work contributes to filling the gap. Qualitative studies can be considered appropriate tools to capture expressive information about beliefs, values, sentiments, and motives10 in understanding the viewpoints of individuals who are overweight and obese. This review of qualitative study aims to map and compile current literature on perceptions of adults with overweight or obesity towards PA and SB, facilitating a comprehensive understanding of their perspectives.\n\n\nMethods\n\nThis systematic review was not pre-registered. We chose Arksey and O’ Malley’s11 framework (2005) for its flexibility, enabling the inclusion of a diverse range of study designs and broad concepts within the systematic review process. We followed the procedural guidelines detailed in the methodology manual provided by Levac et al.12 and quality assessment using Joanna Briggs Institute13 checklist. The preferred reporting items for Systematic Reviews and Meta-analysis Guidelines-Scoping Review guidelines guided this review.14 The checklist details are uploaded in Figshare as extended data document 1.29\n\nThis systematic review focused on research pertaining to perception, behavior, or attitude toward PA and SB among adults with overweight/obesity (Table 1).\n\nAn extensive search was conducted across four electronic databases- Medline via PubMed, Web of Science, Scopus, and CINAHL- employing a varied array of keywords to maximize inclusivity and sensitivity in identifying relevant research published from 2000-2024. The search strategy was developed in consultation with the Information Specialist and subject matter expert (NB, VK, MK, AV, and AKB) through multiple rounds of brainstorming sessions. The final search in all the databases has been conducted by two independent authors (NB and MK). The search term were “overweight,” “obese,” “obesity population,” “adult,” “older adult”, “adult population”, “Physical activity,” “physical inactivity”, “exercise”, “sedentary behaviour”, “sedentary lifestyle”, “perception”, “attitude, and beliefs”, “qualitative studies”, “mixed methods study”, “perception study”. The search strategy was developed with the belief that it would capture all relevant information related to PA and SB perception in adults with overweight/obesity. Apart from electronic searches, manual exploration of the reference lists of all incorporated studies was executed to ensure the inclusion of eligible and pertinent research.\n\nTwo independent reviewers (NB and MK) conducted the eligibility evaluation using Rayyan.qcri.org software.15 A total of 2881 records were identified, wherein 290 records were duplicated and deleted. A systematic title and abstracts (Ti-Ab) screening for 2591 records was conducted by NB and MK based on the predefined criteria. Among the 45 articles selected for full-text screening, nine met the eligibility criteria and were included in the review. Disagreements between the reviewers were resolved through consensus for two studies, with the reviewers (VK, AV, and AKB) stepping in when consensus was difficult to reach. The search and screening procedures, adhering to PRISMA guidelines, are visually depicted in Figure 1.\n\nFrom: Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ 2021;372:n71. doi: 10.1136/bmj.n71.\n\nThe authors (NB, VK, MK, AV, and AKB) prepared a data extraction tool, which underwent pilot testing to ensure relevance. Adjustments were made to the data sheet to meet the review’s objectives. Subsequently, two independent reviewers (NB and MK) manually extracted the data from included studies. Table 2 provides a comprehensive overview of study characteristics for each included study, encompassing details such as authors, publication year, study setting, sample size, and population demographics (age and cultural identification).\n\nEach included study underwent critical appraisal using Joanna Brigg’s Institute critical appraisal tool for Qualitative studies16 by two independent authors (NB and MK). The assessment revealed methodological quality scores surpassing seven (7/10), indicating a good classification. Any discrepancies identified during the quality assessment process were addressed through extensive discussions and mutual agreement among the reviewers. In cases where disagreements persisted, the authors (VK, AV) were consulted to facilitate resolution. The quality assessment of the articles included is summarized in Table 3.\n\n\nResults\n\nA total of nine studies from different countries were included in this scoping review. Perception of PA in adults with overweight/obesity from all nine studies is represented in Table 4, where (+) and (-) indicate positive and negative understanding, respectively.\n\nAdults with overweight/obesity demonstrated a substantial awareness of the benefits of PA and the risks associated with a sedentary lifestyle. Despite this awareness, individuals tended to neglect regular PA, influenced by various factors. Notable contributors to this behavior included a lack of motivation, lack of self-efficacy, time constraints, excessive use of technology, and responsibilities related to work, family, and community. On the positive side, studies also highlighted factors encouraging PA, such as social support, weight loss goals, positive past experiences, overall health benefits, and access to facilities.\n\nFurther detailed findings are framed under the following headings.\n\nAdults consistently link their perceptions of physical activity to both health benefits and physical appearance. They consistently view PA as the primary approach to achieving physical fitness, maintaining mental stability, and reducing the risk of chronic illnesses. Among nine studies, seven studies reported PA needs to be encouraged to ensure living healthy and disease-free17-20,22,24,25. Witnessing friends and family members coping with long-term chronic conditions serves as a significant motivator for them to engage in PA and take charge of their health.20 The relationship between PA and mental health is closely intertwined with mindfulness, happiness, stress reduction, and overall improved state of mind.19,25 A sense of accountability, the role of being providers for the family and community, and the desire to serve as healthy role models for the children became the few most typical reasons for achieving physical fitness.22\n\nAn individual’s attitudes, deeply rooted in personal beliefs, perceptions, and motivators, act as influential factors that can either propel or hinder an individual’s commitment to maintaining an active and healthy lifestyle. Those with a positive attitude or experience view PA as a beneficial and enjoyable pursuit, which significantly influences PA promotion.22 When individuals derive enjoyment and satisfaction and witness positive outcomes such as weight loss from engaging in PA, it contributes to a heightened sense of self-efficacy.22 These positive associations foster a more favorable attitude towards exercise and motivate participants, encouraging sustained participation. Conversely, negative attitudes, such as finding exercise boring, monotony in routine activities, or experiencing dissatisfaction due to lack of perceived benefits, can lead to demotivation and hinder the adoption of regular PA.21\n\nThe present scoping review identified several facilitators and barriers among adults with overweight/obesity concerning PA participation. Commonly reported facilitators included perceived health benefits for oneself and family, social interaction with peers,17,20,22,24,25 weight loss goals,18,20,22,25 improved mental well-being, and increased self-esteem and confidence.22,25 Physical appearance and attractiveness were potential motivators for young men.25 Gender norms were observed to limit opportunities for exercise in women.20 Other barriers identified in this population included lack of safety,21 motivation,17–22,24,25 limited resources, time constraints,17–22,24,25 overuse of technology, fatigue,17,21 laziness, family responsibilities, and shifting cultural and social values. Moreover, increased weight and associated injuries were observed to limit PA among this population.21 Recognizing these factors can help design practical and sustainable approaches to encourage PA in adults with overweight/obesity.\n\nThis review revealed few commonly reported time-saving strategies to promote PA within this population. Practical approaches identified were implementing workplace activities,22 setting specific achievable goals, and incorporating social changes like engaging in activities with friends and family during leisure time.17,20,22,24,25 Moreover, altering social activities to prioritize active pursuits and reduce sedentary time emerged as an important strategy. Participating in gym activities alongside peers may seem appealing; however, its costliness, need for a trainer, and inconvenient timings render it a subjective option for many.24 Also, self-reported interventional approaches towards PA promotion, such as the use of technology where text messages or phone calls could be used as reminders for an individual to move around and also help them to track and compare the process throughout.17 Rewards and reinforcements or gift cards or tangible products (like clothing) were a few incentives reported by women who were overweight.22 Women also mentioned walking, dancing, active jobs, and joining Zumba classes with friends as a few strategies to engage themselves, along with the guilt of missing those classes.20 Social media and public health campaigns were credited for enhancing the knowledge about obesity-related diseases.17\n\nAmong the nine studies, three of them17,21,22 investigated perceptions and attitudes regarding sedentary behavior, as well as the various factors influencing sedentarism. A common theme emerging across these studies are represented under the following headings.\n\nAdults with overweight/obesity often exhibit a lack of awareness regarding sedentarism and its associated risks, primarily stemming from a deficiency in knowledge. However, few adults perceived sedentarism as a prolonged sitting period with minimal movement,23 whereas few perceived it as a risk factor for obesity, various chronic diseases, and other health deteriorating conditions.19 Adults who are overweight often experience SB as a form of relaxation after extended working hours and intertwined with increased leisure time and activities.\n\nThis review identified the contributors to SB, specifically among adults with overweight/obese, categorizing them into personal, social, and environmental factors. Personal factors include daily routine activities such as prolonged periods of inactivity in bed, excessive television viewing, seeking enjoyment and relaxation, allocating personal time, and experiencing reduced productivity post-work. Social factors revolve around social events and the influence of friends and family contributing to SB.21 Both work and home environments play significant roles in influencing SB, with prolonged sitting for relaxation or meeting job demands being prominent factors. Age emerges as a frequent influencer of SB, often leading to decreased PA levels and prolonged sitting.23\n\nIn recent decades, cultural and social lifestyles have significantly changed, leading to a surge in SB characterized by increased sitting and relaxation. The pervasive influence of technology in society has further amplified this trend, resulting in decreased levels of PA.23,25 Additionally, social factors play a crucial role, including the prevalence of desk-bound jobs, leisure time sitting, and heavy reliance on vehicles,23,25 computers, and cell phones at home and in the workplace,23 all contributing substantially to increased sedentary lifestyle in this demographic group.\n\nIt is paramount to implement strategies aimed at breaking sedentary patterns in adults with overweight/obesity to foster healthier lifestyle habits. These strategies include incorporating regular breaks during work hours, integrating PA into daily routines, transforming social activities into more active behaviors, utilizing feedback and prompts as reminders,17,23 and seeking social support to sustain these changes.23 Workplace incentives can play a significant role in enhancing PA behavior and reducing SB. Among these strategies, an initial focus should be on raising awareness about the adverse effects of a sedentary lifestyle. Additionally, addressing the impact of technology-driven SB and implementing diverse approaches to adapt to prevailing work cultures are crucial steps.23,25\n\n\nDiscussion\n\nThis review aimed to gather insights into the perceptions of adults with overweight/obesity regarding PA and SB. By collating diverse perspectives related to PA and SB, our objective was to comprehensively understand the emerging concerns. Key findings emerged across the multiple themes, including factors influencing motivation and limitations to PA, positive and negative attitudes, and various strategies fostering consistent engagement in PA.\n\nCommonly identified facilitators influencing PA in adults with overweight/obesity are health benefits, overall physical fitness, attractive body image, social support, positive past experiences with exercise, weight loss goals, increased self-esteem, and confidence. These factors were similar to a few studies reported in a systematic review conducted in 2021.26 The importance of social support from family members or peers in encouraging and facilitating participation in PA has been highlighted, similar to the findings of a review conducted in 2023.27 The social aspect of PA makes it more feasible and enjoyable and promotes regular engagement. This review also reports barriers that hinder PA, like time constraints, lack of motivation, lack of interest, fear of injuries, increased weight, societal judgment, familial obligations, negative previous experiences, and occupational responsibilities.\n\nA significant proportion of adults with overweight lack familiarity with the term “sedentary,” associating it with prolonged sitting, low PA, relaxation, and leisure pursuits.23 O’Donoghue et al.’s28 2016 systematic review examined factors related to SB in adults similar to this review study, including reading, driving, work-related sitting, cell phone use, and television watching. SB was linked to perceived fatigue and stress while inversely related to perceived health benefits. This review identified urbanization, technological advancements, and increased reliance on motorized work as key drivers for SB, compounded by prolonged periods of sitting during leisure activities or due to work demand. Awareness of associated health risks and effective intervention strategies remains lacking, necessitating attention from researchers and clinicians.\n\nMost studies on this topic have been carried out in developed countries, emphasizing the impact of modernization and mechanization on lifestyle factors. Conversely, research conducted in developing nations has concentrated on issues such as limited access to exercise equipment and facilities, as well as a lack of social support. Safety concerns, particularly among women in developing countries, have also been highlighted.21 Studies have observed a trend toward increased use of technology among young adults, which correlates with reduced PA and a more sedentary lifestyle.24,25 Across all studies, a common theme emerges the necessity of social or peer support and adopting time-efficient approaches to promote physical activity.\n\nSeveral studies have delved into the multifaceted factors influencing obesity, focusing on diet, food habits, and nutrition alongside PA. In a study in 2015, it was reported that some adults who were overweight perceived healthy nutrition as a potential alternative to PA.20 Additionally, research has identified various obstacles to adopting healthy eating habits, such as insufficient knowledge about nutritious foods, challenges in preparing healthy meals, and the pervasive availability of unhealthy food options.21,25 Moreover, an unsupportive social environment has been highlighted as a significant barrier to maintaining a healthy diet.21,25 Particularly, women often face dilemmas regarding whether to prioritize purchasing healthier albeit more expensive food or opting for cheaper, less nutritious alternatives.21\n\nThis systematic review benefits from its comprehensive screening of a substantial number of abstracts and articles, providing an extensive mapping of perceptions regarding PA and SB among adults with overweight/obesity. This systematic review focuses exclusively on qualitative studies, offering valuable insights into subjective experiences. Additionally, the review assessed the quality of articles, identifying high-quality materials for inclusion, thus ensuring rigor in the analysis. However, this study has a few limitations as the quantitative aspect of the topic was not analysed. Furthermore, researcher bias may influence the interpretation of the findings, which may limit their broader applicability.\n\nA range of attitudes influencing the willingness and unwillingness of adults with overweight/obesity to engage in PA were uncovered, which highlights the importance of addressing psychological barriers and promoting positive self-perception as part of efforts to promote PA. Strategies to promote PA and reduce SB were identified, offering a roadmap for interventions to foster engagement and adherence among this population. Leveraging technology to improve adherence to regular PA (e.g., virtual coaching platforms, goal-setting mechanisms using fitness trackers and apps) can be explored in further research.\n\n\nConclusion\n\nThe intricate and diverse perspectives held by adults with overweight/obesity, particularly concerning their PA and SB, offer valuable insights for clinicians and researchers aiming to promote regular PA. Additionally, the study results address the unique challenges faced in workplace environments when adopting sustainable PA. Implementing cost-effective approaches is equally imperative to make interventions accessible and feasible for individuals dealing with overweight/obesity.", "appendix": "Data availability statement\n\nAll data underlying the results are available as part of the article, and no additional source data are required.\n\nReporting guidelines\n\nFigshare: Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist for Physical Activity and Sedentary Behavior Perceptions in Overweight and Obese Adults: A Qualitative Review; Figure. https://doi.org/10.6084/m9.figshare.25997038.v1. 29\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe express our gratitude to Manipal Academy of Higher Education, Manipal, for furnishing the essential resources necessary for the successful completion of our research.\n\n\nReferences\n\nEllulu MS, Patimah I, Khaza’ai H, et al.: Obesity and inflammation: the linking mechanism and the complications. Arch. Med. Sci. 2017; 13(4): 851–863. PubMed Abstract \| Publisher Full Text\n\nObesity and overweight: 2009. Reference Source\n\nObesity and overweight: 2024. Reference Source\n\nBoutari C, Mantzoros CS: A 2022 update on the epidemiology of obesity and a call to action: as its twin COVID-19 pandemic appears to be receding, the obesity and dysmetabolism pandemic continues to rage on. Metabolism. 2022; 133: 155217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGBD 2015 Obesity CollaboratorsAfshin A, Forouzanfar MH, et al.: Health Effects of Overweight and Obesity in 195 Countries over 25 Years. N. Engl. J. Med. 2017; 377(1): 13–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institute of Child Health and Human Development. Reference Source\n\nTudor-Locke C, Brashear MM, Johnson WD, et al.: Accelerometer profiles of physical activity and inactivity in normal weight, overweight, and obese U.S. men and women. Int. J. Behav. Nutr. Phys. Act. 2010; 7: 60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSedentary Behaviour Research Network: Letter to the editor: standardized use of the terms “sedentary” and “sedentary behaviours”. Appl. Physiol. Nutr. Metab. 2012; 37(3): 540–542. Publisher Full Text\n\nHansen BH, Holme I, Anderssen SA, et al.: Patterns of objectively measured physical activity in normal weight, overweight, and obese individuals (20-85 years): a cross-sectional study. PLoS One. 2013; 8(1): e53044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerkwits M, Inui TS: Making use of qualitative research techniques. J. Gen. Intern. Med. 1998; 13(3): 195–199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArksey H, O’Malley L: Scoping studies: towards a methodological framework. Int. J. Soc. Res. Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nLevac D, Colquhoun H, O’Brien KK: Scoping studies: advancing the methodology. Implement. Sci. 2010; 5: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters MDJ, Marnie C, Tricco AC, et al.: Updated methodological guidance for the conduct of scoping reviews. JBI Evid. Synth. 2020; 18(10): 2119–2126. Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann. Intern. Med. 2018; 169(7): 467–473. PubMed Abstract \| Publisher Full Text\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan-a web and mobile app for systematic reviews. Syst. Rev. 2016; 5(1): 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoanna Briggs Institute: Critical Appraisal Tools.Reference Source\n\nGarcia DO, Valdez LA, Hooker SP: Hispanic Male’s Perspectives of Health Behaviors Related to Weight Management. Am. J. Mens Health. 2017; 11(5): 1547–1559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuess N: A qualitative investigation of attitudes towards aerobic and resistance exercise amongst overweight and obese individuals. BMC. Res. Notes. 2012; 5: 191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarua S, Saikia N: Perception, risk factors, and health behaviours in adult obesity in Kolkata, India: a mixed methods approach. BMC Public Health. 2022; 22(1): 2376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvarado M, Murphy MM, Guell C: Barriers and facilitators to physical activity amongst overweight and obese women in an Afro-Caribbean population: A qualitative study. Int. J. Behav. Nutr. Phys. Act. 2015; 12: 97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaruth M, Sharpe PA, Parra-Medina D, et al.: Perceived barriers to exercise and healthy eating among women from disadvantaged neighborhoods: results from a focus groups assessment. Women Health. 2014; 54(4): 336–353. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoseph RP, Ainsworth BE, Mathis L, et al.: Utility of Social Cognitive Theory in Intervention Design for Promoting Physical Activity among African-American Women: A Qualitative Study. Am. J. Health Behav. 2017; 41(5): 518–533. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarren TY, Wilcox S, St George SM, et al.: African American Women’s Perceived Influences on and Strategies to Reduce Sedentary Behavior. Qual. Health Res. 2018; 28(7): 1112–1122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarbrick I, Wilson D, Boulton A: Provider, father, and bro--Sedentary Māori men and their thoughts on physical activity. Int. J. Equity Health. 2016; 15: 22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshton LM, Hutchesson MJ, Rollo ME, et al.: Young adult males’ motivators and perceived barriers towards eating healthily and being active: a qualitative study. Int. J. Behav. Nutr. Phys. Act. 2015; 12: 93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaillot A, Chenail S, Barros Polita N, et al.: Physical activity motives, barriers, and preferences in people with obesity: A systematic review. PLoS One. 2021; 16(6): e0253114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurran F, Davis ME, Murphy K, et al.: Correlates of physical activity and sedentary behavior in adults living with overweight and obesity: A systematic review. Obes. Rev. 2023; 24(11): e13615. PubMed Abstract \| Publisher Full Text\n\nO’Donoghue G, Perchoux C, Mensah K, et al.: A systematic review of correlates of sedentary behaviour in adults aged 18-65 years: a socio-ecological approach. BMC Public Health. 2016; 16: 163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinha MK: Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist for Physical Activity and Sedentary Behavior Perceptions in Overweight and Obese Adults: A Qualitative Review. figshare. Figure. 2024. Publisher Full Text" }	[ { "id": "302707", "date": "07 Aug 2024", "name": "Saumya Srivastava", "expertise": [ "Reviewer Expertise Sacroiliac joint dysfunction", "Low back pain", "Mckenzie", "Mulligan" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nArticle Summary The article \"Physical activity and sedentary behaviour perceptions in overweight and obese adults: A systematic review of qualitative study\" by Bora N, K V, Verma A et al., examines the perceptions of physical activity and sedentary behaviour in overweight and obese adults. The authors conducted a systematic review of qualitative studies to understand the barriers and facilitators influencing physical activity and sedentary behaviours in this population.\nFindings: 1. Barriers to Physical Activity: Overweight and obese adults reported several barriers, including physical limitations, lack of motivation, and psychological factors such as body image issues and fear of judgement. 2. Facilitators to Physical Activity: Support from family, friends, and healthcare providers, along with the availability of suitable environments and resources, were identified as key facilitators. 3. Perceptions of Sedentary Behaviour: Many participants viewed sedentary behaviour as a habitual and difficult-to-change aspect of their lives, often associated with relaxation and leisure activities. 4. Intervention Preferences: The participants expressed a preference for personalised, enjoyable, and non-judgmental physical activity interventions.\nStrengths and Weaknesses\nStrengths 1. Comprehensive Review: The systematic review includes a wide range of qualitative studies, providing a broad understanding of the perceptions of physical activity and sedentary behaviour in overweight and obese adults. 2. Depth of Insights: The qualitative nature of the included studies allows for in-depth insights into the personal experiences and perspectives of the participants. 3.Identification of Key Factors: The study effectively identifies and categorises the barriers and facilitators to physical activity, which can inform the design of targeted interventions. 4. Practical Implications: The findings have practical implications for healthcare providers in designing and implementing effective physical activity programs for overweight and obese individuals.\nWeaknesses 1. Heterogeneity of Studies: The included studies vary in terms of their methodologies, participant demographics, and contexts, which may affect the generalizability of the findings. 2. Publication Bias: As the review focuses on published qualitative studies, there may be a publication bias, with positive findings more likely to be published than negative or null results. 3.Lack of Quantitative Data: The reliance on qualitative data limits the ability to quantify the impact of identified barriers and facilitators on physical activity levels. 4.Potential for Subjectivity: The interpretation of qualitative data can be subjective, and the review's findings may be influenced by the authors' perspectives and biases.\nRelevance and Positive Comments The article is highly relevant for researchers and healthcare providers interested in promoting physical activity among overweight and obese adults. By providing a comprehensive overview of the barriers and facilitators, the study highlights critical areas for intervention and support.\nPositive Comment: The systematic review by Bora et al. is a valuable contribution to the field of public health and physical activity research. Its in-depth analysis of qualitative studies provides nuanced insights into the lived experiences of overweight and obese adults, offering a robust foundation for designing effective, empathy-driven interventions to promote physical activity and reduce sedentary behaviour in this population.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] }, { "id": "302702", "date": "09 Aug 2024", "name": "Dr Doss Prakash S", "expertise": [ "Reviewer Expertise Community Health", "Public Health", "Geriatrics", "Women's Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe systematic review have highlighted multifaceted factors leading to overweight and obesity in adult population. This review will be an valuable clinical tool for the health care professionals for clinical decision making process. And also provides convincing evidence to promote physical activity among the over weight and obese adults to prevent any medical complications.\n\nReview point: The overall article is in line with systematic review findings, but it requires some revision…\n\nAuthors need to be specific in the introduction section about the literature gap… currently known and the clinical relevance point Include the prevalence of physical activity inactivity as reported in the current WHO report. As the article is about overweight and obese adults, therefore author needs to justify the need for study for both groups (overweight and obese adults) in the introduction section Were there any specific findings reported in the published literature concerning the inclusion criteria of ages 18 to 60 years in the study? Highlight the key findings. Report the characteristics of the included study, specifically focusing on socioeconomic status, occupation, and behavioral information if it is provided in the paper. If this information is not included, discuss its potential impact. Any specific information related to digital, gender, or age group? Is there any information about readiness to change perception? PA perception with health benefits- this section there is one line …. The relationship between PA and mental health is closely intertwined with mindfulness, happiness, stress reduction, and overall improved state of mind- need clarity. Also, in the same section (PA perception with health benefits), what does the adoption of regular PA mean?\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-787
https://f1000research.com/articles/10-350/v1	06 May 21	{ "type": "Systematic Review", "title": "Factors related to loss of appetite in postoperative cardiac surgery patients: A systematic review", "authors": [ "Channarong Prasankok", "Samoraphop Banharak", "Channarong Prasankok" ], "abstract": "Background: Postoperative cardiac surgery patients often experience appetite loss. Although nutritional status is known to be associated with time of recovery, functional status, and length of stay, less is known about factors related to patient’s loss of appetite after cardiac surgery. This review aimed to identify and understand factors related to loss of appetite in postoperative cardiac surgery patients, systematic review with narrative summary design was applied. Data sources including CINAHL, SCOPUS, PubMed, ProQuest, ScienceDirect, ThaiLIS, ThaiJo, and E-Thesis were searched without restriction on publication year through August 2020. Methods: We conducted the review following the Joanna Briggs Institute methodology, using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist to categorize methodological quality and the PRISMA flow diagram to record the studies’ factors. Results: Six studies reported one or more of 16 factors related to loss of appetite: older age, sex (female), illiteracy, history of chronic disease, not knowing someone in health field, pain score ≥ 7, pain medications containing codeine, constipation, depression, heart-lung machine ≥ 120 minutes, preoperative serum creatinine levels ≥ 179 µmol/L, emergency surgery, perfusion pressure ≤ 40 mmHg, low cardiac output syndrome, mechanical ventilation ≥ 96 hours, and a New York Heart Association class III and IV. Conclusion: The small number of publications restrict our conclusions. Future research should focus on multiple factors related to appetite loss in postoperative cardiac surgery patients. Additional research will provide a foundation for evidence-based interventions to reduce appetite loss and improve patient nutritional status after cardiac surgery. Nurses and other health professionals should assess postoperative cardiac surgery patients for the presence of the 16 significant factors. To promote patients’ nutritional status, there should be evidence-based practice guidelines on the management of postoperative symptoms such as pain management, treatment of constipation, and reduction of emotional stress and depression.", "keywords": [ "anorexia", "cardiac surgery", "heart surgery", "loss of appetite", "poor appetite", "systematic review" ], "content": "Introduction\n\nHeart disease is one of the leading causes of death in both developed and developing countries1. Although treatment depends on severity and types of heart disease, first line approaches include lifestyle changes involving nutrition, exercise, and medication. As patients’ symptoms worsen or complications increase, cardiac surgery often becomes the next treatment of choice1.\n\nThere are various types of cardiac surgery. These include coronary artery bypass graft, valve replacement and repair, great vessel surgery, and septum repair for those with congenital heart disease. Most of these surgeries require the use of the heart-lung machine as a cardiopulmonary bypass to take over the function of the heart and lungs during surgery to help surgeons repair a diseased heart’s malfunctioning. Because there have been adverse effects from using the heart-lung machine on the various body systems, an off-pump coronary artery bypass technique was developed as an alternative2. Nevertheless, the cardiopulmonary bypass remains widely used during open heart surgery. Prevention and management of possible deleterious effects from its use require ongoing assessment.\n\n\nBackground\n\nAlterations in postoperative physiological/psychological functioning contribute to loss of appetite. Surgery and the use of the heart-lung machine cause injuries in tissues and peripheral nerves that can lead to a nociceptive and inflammatory response, causing prostaglandins, bradykinins, and substance P to become increased that produces physical pain3. Postoperative pain and the use of the heart-lung machine are stressors that produce a physiological response such as the systemic inflammatory response syndrome3. The syndrome is a disturbance in the balance of the coagulation system that decreases the production of platelets and fibrinogen with increases in prothrombin time, leading to a higher risk of postoperative bleeding4. The immune system responds by decreasing the polymorphonuclear neutrophils and also macrophages in the lungs, which heighten the risk of infections, including pneumonia5. Cardiac surgery also intensifies catabolism and insulin resistance, resulting in higher blood sugar levels in both patients with and without a history of diabetes6. These factors directly and indirectly disturb the postoperative physiological/psychological interconnections to produce a loss of appetite and a nutritional imbalance7.\n\nFood consumption behaviors change when patients consume small volumes of food, coupled with physical decline, less body movement/mobility, depression, and postoperative stress. Ścisło et al. (2019) found that after the cardiac surgery, 50% of patients had loss of appetite with an increased risk of poor nutrition, 35.4% had postoperative complications, and 10% had a decreased body mass index (BMI)8. The study affirmed that a decrease in BMI after surgery is associated with postoperative complications9. Chermesh et al. (2014) found similarly that poor nutritional status increases the severity and number of complications in postoperative patients10. Ringaitienė et al. (2016) reported that recovery is delayed in patients who have a loss of appetite and consume less food, causing longer hospitalizations11. Based on clinical experiences in caring for patients who had undergone cardiac surgery, we observed that some patients had decreased appetite, difficulty eating, ate less or had early satiety, and suffered bouts of nausea and vomiting. We further observed that these same postoperative cardiac surgical patients tended to have a late recovery, longer duration of hospitalization, and poorer prognosis than others without the symptoms. To the best of our knowledge, we found no literature that summarized and explained our observations. Therefore, the purpose of this systematic review of the literature was to contribute to a body of knowledge by focusing on all known factors related to loss of appetite in postoperative cardiac surgery patients. Understanding these factors will provide a foundation that is necessary to support nursing practice of cardiac surgical patients and inform future researchers what further aspects of loss of appetite need to be studied.\n\n\nPurpose\n\nThe purpose of this review was to systematically identify and understand the collective factors related to loss of appetite in postoperative cardiac surgery patients as found in published research.\n\n\nMethods\n\nWe followed the systematic review process with narrative summary as prescribed by the Joanna Briggs Institute (JBI) methodology by formulating the review question, defining the inclusion and exclusion criteria, developing the search strategy, locating and selecting studies, assessing their quality, extracting data, and analyzing and interpreting the results12. Moreover, the protocol for systematic review was registered at PROSPERO on February 1, 2021 and registration number was CRD42021234615.\n\nWe searched CINAHL, SCOPUS, PubMed, ProQuest, ScienceDirect, ThaiLIS, ThaiJo, and E-Thesis databases and used snowballing through reference lists of defined studies. The search for both English and Thai published papers was made without restriction on publication year through August 2020. The search strategy used the keywords “cardiac surgery” OR “open-heart surgery” AND “poor appetite” OR “loss of appetite” OR “decreased appetite” OR “change in appetite” OR “anorexia.” The selection of studies was made based on the following inclusion criteria: (a) research in humans related to cardiac surgery; (b) results about factors related to loss of appetite in postoperative cardiac surgery patients; (c) patients aged 18 years and older; and (d) published in the English or Thai language.\n\nThere were 5,774 original studies from the databases and two from hand searches. The winnowing process began with deduplication, which left 3,320 studies. Only 74 studies had full-text availability, of which 68 were excluded because they did not fully meet the inclusion criteria. As a result, six empirical research articles remained for appraisal. Figure 1 displays the PRISMA flow diagram of the information flow during the review process.\n\nWe individually reviewed the six selected articles using the standardized critical appraisal instrument from the JBI for analytical cross-sectional studies13. The studies were required to meet a positive response (i.e., “yes”) on a minimum of four of the eight questions on the critical appraisal tool. Methodological quality was grouped into the four categories of very low, low, moderate, and high quality.\n\nOn the JBI data extraction form (Aromataris & Munn, 2018)12, we recorded the studies’ authors, study designs, settings, participants, levels of evidence certainty and methodological quality, types of surgery, times of outcomes measuring, and factors related to loss of appetite. Prior to starting the review, we practiced article screening, data extraction, and quality assessment with two other researchers who independently verified that the process and results we had undertaken were accurate. If there were incongruent opinions during the actual review process, we reached consensus in mutual discussion.\n\nExtraction of quantitative data to conduct meta-analysis was not possible due to the heterogeneity of the study population, different types of cardiac surgery, outcome measures, and data analysis across the studies. The findings have been presented and discussed in tabular and narrative form to aid the data presentation.\n\n\nResults\n\nSix research articles met the inclusion criteria14–19. They were all descriptive studies. Five had a moderate level of quality, and one had a high level of quality (Table 1 & Table 2). Two studies were published before the year 200016,17, and four were published on or after 200014,15,18,19. Patients in five studies had undergone coronary artery bypass graft surgery14,16–19; patients in three studies had undergone valve surgery15,16,19; and patients in one study had both a septum repair for congenital heart disease and a repair of the aorta19. The surgeries reported in five studies required cardiopulmonary bypass14–16,18,19, whereas one study did not specify its use17. Although five studies addressed patients postoperatively and after-discharge14–18, one study described the preoperative, operative, and postoperative phases19 (Table 3).\n\na = Risk of bias; b = Precision; c = Directness; d = Consistency\n\nGRADE Working Group Grades of Evidence for certainty of evidence\n\nHigh: The research provides a very good indication of the likely effect. The likelihood that the effect will be substantially different is low.\n\nModerate: The research provides a good indication of the likely effect. The likelihood that the effect will be substantially different is moderate.\n\nLow: The research provides some indication of the likely effect. The likelihood that it will be substantially different (a large enough difference that it might have an effect on a decision) is high.\n\nVery low: The research does not provide a reliable indication of the likely effect. The likelihood that the effect will be substantially different (a large enough difference that it might have an effect on a decision) is very high.\n\n†The scores of Methodological Quality of the Studies are shown in fractions based on Joanna Briggs Institute and the Mixed Methods Appraisal Tools.\n\n⊕◯◯◯ = Very low quality; ⊕⊕◯◯ = Low quality; ⊕⊕⊕◯ = Moderate quality; ⊕⊕⊕⊕ = High quality\n\nThere were 16 factors identified in the six studies related to loss of appetite and they were divided into three phases. The preoperative factors were (a) age ≥ 60 years old; (b) sex (female); (c) history of a chronic disease, such as diabetes, hypertension, hyperlipidemia; (d) illiteracy; (e) not knowing someone in the health field; and (f) preoperative serum creatinine level ≥ 179 µmol/L. The operative factors were (a) emergency surgery; (b) using the heart-lung machine ≥ 120 minutes; (c) perfusion pressure ≤ 40 mmHg; and (d) low cardiac output syndrome. Finally, the postoperative factors were (a) using mechanical ventilation ≥ 96 hours; (b) moderate or higher pain score (≥ 7 points); (c) using pain medications containing codeine; (d) constipation; (e) depression; and (f) New York Heart Association (NYHA) class III and IV. Nevertheless, the study of Zang et al. (2009)19 reported specifically that the patient’s sex and types of cardioplegia solutions had no impact on loss of appetite in postoperative cardiac surgery patients (Table 3).\n\n\nDiscussion\n\nWe found that there were 16 factors related to loss of appetite in cardiac surgery patients. They were present in the preoperative, operative, or postoperative/discharge phases.\n\nThe aging process in itself can produce a loss of appetite due to a decrease in taste and smell, resulting in discontent with eating and a poor appetite20. Older people (≥ 60 years) experience a decrease in ghrelin or a hunger hormone, an increase in leptin and insulin, and changes in the gastrointestinal system and inflammatory process, such as an increase in interleukin 1 (IL-1) and 6 (IL-6) and tumor necrosis factor alpha (TNF-α)21 that contribute to a loss of appetite. Older females, with low estrogen levels transitioning through menopause or post menopause can also have a loss of appetite20. Whether males or females are more likely to have a loss of appetite with cardiac surgery, however, needs further study, since Zang et al. (2009) found there was no statistical difference between the two19. Chronic disease, such as diabetes or hypertension, can lead to an inflammatory process and an increase in interleukin and tumor necrosis factor alpha, which are known to be related to appetite22,23. Moreover, increased blood sugar and high blood pressure can produce physiological stress. Both may directly and indirectly cause loss of appetite by pathophysiological and psychological pathways7.\n\nIlliterate patients and those who do not know someone in the health field tend to experience loss of appetite. They may be less knowledgeable and unsure of self-care activities or uncomfortable and inexperienced in seeking help with their health and nutrition problems. Cebeci and Celik (2008) and Choocherd et al. (2016) also found that low health literacy was related to nutritional problems in coronary artery bypass graft surgery patients24,25. High preoperative serum creatinine also affects loss of appetite, but indirectly24,25. A decline in glomerular filtration is associated with reduced food intake26–28. Although hospitals are not traditionally known for tasty cuisine, prolonged lengths of stay in an intensive care unit can produce depression that may lead to a lack of appetite26,29,30.\n\nDuring the use of the heart-lung machine, chemical substances are produced by a systemic inflammatory response named proinflammatory cytokine31. This substance includes interleukin (IL), tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ) and granulocyte-macrophage colony stimulating factor (GM-CSF). Higher levels of chemical substances are strongly related to loss of appetite. These chemical substances also become substantially increased in patients with cancer, affecting appetite. After the heart-lung machine is disconnected, the substances can remain at high levels for 48 hours after surgery32. Not only does the heart-lung machine produce a systemic inflammatory response, but the digestive system is interrupted. Prolonged use of the heart-lung machine can lead to weariness and loss of appetite after cardiac surgery32.\n\nLow cardiac output syndrome, low perfusion pressure, and emergency surgery may indirectly affect loss of appetite. These three factors correlate with extended stays in the intensive care unit, total lengths of hospitalization, and prolonged intubation times26,28. Low cardiac output and low perfusion pressure place patients at risk of acute kidney injury that can lead to high serum creatinine; and emergency cardiac surgery causes more postoperative complications in patients than planned surgery26,27,33. These three conditions may collectively bring about stress and depression, leading to loss of appetite26,29,30,34.\n\nProlonged mechanical ventilation requires suctioning patients’ endotracheal tubes to clear the airway, leading to post-extubation fatigue and loss of appetite. It is a significant factor affecting patients’ appetite levels after their endotracheal tubes are removed. Longer periods of postoperative mechanical ventilation are also influenced by higher levels of preoperative creatinine, low cardiac output, and psychological depression, further contributing to loss of appetite35. Depression itself is a source of appetite loss by the hypoactivation of the brain’s insular regions, especially mid-insular cortex that supports the body’s physiological control of eating36. Depressed patients have an increase in their hypothalamic-pituitary-adrenal drive that can cause high levels of cortisol, which is associated with decreased appetite and hypoactivation of food-motivation neurocircuitry30,34,37. The interactivity and interaction among these regions, however, contribute to individual differences in patients with depression-related appetite changes36.\n\nAfter open heart surgery, patients experience pain and discomfort during routine postoperative care, such as deep breathing and coughing to expand the lungs and clear secretions, using an incentive spirometer, moving/turning in bed coupled with early ambulation, and undergoing physical therapy with possible rehabilitation activities. Pain decreases the movement and mobility of patients, so the stomach and intestine are relatively less functional38. Patients can feel anxious, insecure, and uncertain about the disease and success of the surgery, causing them to have emotional stress. These complicated reactions are intercorrelated and can affect the appetite directly and indirectly. As a result, these patients may experience a decrease in appetite with a distorted perception of smell and gustatory dysfunction, leading to a lower consumption of food39.\n\nPain that occurs immediately after cardiac surgery is called acute pain, whereas pain after hospital discharge is chronic pain. Both can stimulate the inflammatory process, which causes the release of cytokine, especially IL-640,41. Interleukine-6 is related to loss of appetite. Because the release of cytokine leads to the pain cycle, postoperative patients require analgesics, usually with codeine42,43. Medications containing codeine coupled with less physical movement and stomach/intestinal dysfunction may cause constipation. Constipation affects loss of appetite because patients feel that food remains in the gastrointestinal system, which produces abdominal discomfort38,44.\n\nPatients with NYHA Class III & IV heart failure may have a loss of appetite due to hypervolemia, especially if there is congestion in the lungs, liver, and intestine45. Congestion directly affects the function of the respiratory and gastrointestinal systems, producing dyspnea, fatigue, and inactivity, all related to loss of appetite. Liver and intestinal congestion leads to gastrointestinal hypomotility of the stomach and intestine46. This may cause abdominal distension and discomfort, thus inhibiting patient activities would stimulate appetite. In addition, medications that treat and control heart failure, such as digoxin and simvastatin, not only impact appetite indirectly by affecting the function of the gastrointestinal system but also decrease appetite directly46,47.\n\nFive out of the six studies had a moderate level of methodological quality14,16–19. The researchers did not report their confounding factors, missing data, or how they managed the statistical challenges. Only one study (Corrêa & Cruz, 2000) reported controlling for two confounding factors that may have affected loss of appetite (i.e. level of pain and onset of having pain)15. Pain is both a rapidly (state variable) and slowly (trait variable) changing factor48,49. A state variable can change in a short time period and at a specific moment, whereas a trait variable takes much longer to change. Corrêa and Cruz (2000) divided the samples into several groups based on the level and onset of pain and compared the differences of loss of appetite between each group to identify more clearly the factors related to loss of appetite15. Because only one of the six studies demonstrated high methodological quality by controlling for confounding factors, we cannot make firm conclusions on the overall results of the systematic review12.\n\nSome of the 16 factors we report are not exclusive to postoperative cardiac surgery patients. Other types of surgery can produce changes in eating behaviors, food intake, loss of appetite, and malnutrition in postoperative patients. Factors may include older age50,51; sex (female)50–52; type of illness or chronic disease, such as type 2 diabetes mellitus50,52; medications50; length of hospital stay50; and psychological factors, such as stress, depression, psychopathology, and well-being53. In addition to those reported for general surgery patients, our systematic review expanded knowledge of other specific factors related to loss of appetite in postoperative cardiac surgery patients.\n\n\nStrengths and limitations\n\nWe searched extensively the published literature in eight national and international databases, restricting the studies to those in English or Thai. Out of a large potential number of studies, only six met the specific criterion related to loss of appetite in postoperative cardiac surgery patients14–19. None of the studies had loss of appetite as a primary research objective. Methodological problems in some studies could have been strengthened by having larger sample sizes, reporting how missing data were handled, and mentioning how confounding factors were controlled14,16–19. In the studies with a larger sample size, subgroup analysis on individual factors could have brought better understanding of their influence on loss of appetite19. None of the six studies used an experimental research design and long-term evaluation.\n\n\nConclusions and implication\n\nThis systematic review54 provides initial knowledge for the development of nursing interventions based on the factors related to loss of appetite in postoperative cardiac surgery patients. Clinical assessment by nurses and other health professionals should include whether patients were on the heart-lung machine ≥ 120 minutes, had emergency surgery, reported having a low cardiac output syndrome, or required mechanical ventilation ≥ 96 hours. To promote patient’s nutritional status, there should be evidence-based practice guidelines on the management of postoperative symptoms related to loss of appetite. These would include pain management, treatment of constipation, and reduction of emotional stress and depression. Additional information on self-care should be given to patients and families before hospital discharge to relieve their stress and depression, enhance knowledge of nutrition to promote healing, and prevent loss of appetite. However, nurses and health professionals should first assess health literacy to ensure that patients and families adequately understand the discharge instructions. Discharge planning should provide instruction on how to support self-care ability and reduce post-discharge problems, especially guidance in nutrition after coronary artery bypass graft surgery. A multidisciplinary approach, including physician, nurse, dietitian/nutritionist, and pharmacist, should be considered as part of the holistic care given to postoperative cardiac surgery patients because loss of appetite can result from multiple factors that lead to poor nutrition and inhibit healing.\n\nFuture research should focus on the identified factors related to appetite loss in postoperative cardiac surgery patients. The use of multiple research methods and longitudinal studies will provide a foundation for evidence-based interventions to reduce loss of appetite and improve nutritional status for patients after cardiac surgery.\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 ‘Factors related to loss of appetite in postoperative cardiac surgery patients: A systematic review’, https://doi.org/10.6084/m9.figshare.1447318854\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 are very thankful to those who contributed to this systematic review for their collaboration and support. Special appreciation is extended to Dr. Andrew C. Mills for reviewing early drafts.\n\n\nReferences\n\nAmerican Heart Association: Heart disease and stroke statistics 2019 update: A report from the American Heart Association. Circulation. 2019; 139(10): e56–e528. PubMed Abstract \| Publisher Full Text\n\nCohn LH, Adams DH: Cardiac surgery in the adult (5th edition). McGraw Hill Education, 2016. Reference Source\n\nZubrzycki M, Liebold A, Skrabal C, et al.: Assessment and pathophysiology of pain in cardiac surgery. J Pain Res. 2018; 11: 1599–1611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurphy GJ, Angelini GD: Side effects of cardiopulmonary bypass: What is the reality? J Card Surg. 2004; 19(6): 481–488. PubMed Abstract \| Publisher Full Text\n\nGaudriot B, Uhel F, Gregoire M, et al.: Immune dysfunction after cardiac surgery with cardiopulmonary bypass: Beneficial effects of maintaining mechanical ventilation. Shock. 2015; 44(3): 228–233. PubMed Abstract \| Publisher Full Text\n\nKnapik P, Nadziakiewicz P, Urbanska E, et al.: Cardiopulmonary bypass increases postoperative glycemia and insulin consumption after coronary surgery. Ann Thorac Surg. 2009; 87(6): 1859–1865. PubMed Abstract \| Publisher Full Text\n\nKhamsiri N, Wanitkun N, Sindhu S, et al.: The relationship between blood loss, systemic inflammatory response syndrome, blood glucose, pain, and recovery in patients during the first 12 hours after open heart surgery. Thai Journal of Cardio-Thoracic Nursing. 2012; 23(1): 2–18. Reference Source\n\nŚcisło L, Bizoń A, Walewska E, et al.: Nutritional status of elderly patients after coronary artery bypass surgery. Int J Environ Res Public Health. 2019; 16(2): 226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao M, Sun J, Young N, et al.: Impact of Body Mass Index on Outcomes in Cardiac Surgery. J Cardiothorac Vasc Anesth. 2016; 30(5): 1308–1316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChermesh I, Hajos J, Mashiach T, et al.: Malnutrition in cardiac surgery: Food for thought. Eur J Prev Cardiol. 2014; 21(4): 475–483. PubMed Abstract \| Publisher Full Text\n\nRingaitienė D, Gineitytė D, Vicka V, et al.: Preoperative risk factors of malnutrition for cardiac surgery patients. Acta Med Litu. 2019; 23(2): 99–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAromataris E, Munn Z: Joanna Briggs Institute Reviewer’s Manual. 2018.\n\nSchünemann H, Brożek J, Guyatt G, et al.: Handbook for grading the quality of evidence and the strength of recommendations using the GRADE approach. GRADE handbook (gradepro.org). 2020. Reference Source\n\nAmmouri AA, Al-Daakak ZM, Isac C, et al.: Symptoms experienced by Jordanian men and women after coronary artery bypass graft surgery. Dimens Crit Care Nurs. 2016; 35(3): 125–132. PubMed Abstract \| Publisher Full Text\n\nCorrêa CG, Cruz DA: Pain: Clinical validation with postoperative heart surgery patients. Nurs Diagn. 2000; 11(1): 5–14. PubMed Abstract \| Publisher Full Text\n\nGrap MJ, Savage L, Ball GB: The incidence of gastrointestinal symptoms in cardiac surgery patients through six weeks after discharge. Heart Lung. 1996; 25(6): 444–450. PubMed Abstract \| Publisher Full Text\n\nKing KB, Parrinello KA: Patient perceptions of recovery from coronary artery bypass grafting after discharge from the hospital. Heart Lung. 1998; 17(6 Pt 1): 708–15. PubMed Abstract\n\nMiller KH, Grindel CG: Comparison of symptoms of younger and older patients undergoing coronary artery bypass surgery. Clin Nurs Res. 2004; 13(3): 179–93; discussion 194-8. PubMed Abstract \| Publisher Full Text\n\nZhang G, Wu N, Liu H, et al.: Case control study of gastrointestinal complications after cardiopulmonary bypass heart surgery. Perfusion. 2009; 24(3): 173–178. PubMed Abstract \| Publisher Full Text\n\nMuangpaisan W (Ed.): Gerontology and geriatrics for primary care practice. Thai Society of Gerontology and Geriatric Medicine. 2017.\n\nLandi F, Calvani R, Tosato M, et al.: Anorexia of aging: Risk factors, consequences, and potential treatments. Nutrients. 2016; 8(2): 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalassetti P: Inflammation and oxidative stress in obesity, metabolic syndrome, and diabetes. Exp Diabetes Res. 2012; 2012: 943706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansson GK: Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352(16): 1685–1695. PubMed Abstract \| Publisher Full Text\n\nCebeci F, Celik SS: Discharge training and counselling increase self-care ability and reduce postdischarge problems in coronary artery bypass graft surgery patients. J Clin Nurs. 2008; 17(3): 412–420. PubMed Abstract \| Publisher Full Text\n\nChoocherd S, Wanitkun N, Danaidusadeekul S, et al.: Relationships among health literacy, severity of disease, patient engagement, perception of person-centered care and quality of life in patients after coronary artery bypass graft surgery. Journal of Nursing Science. 2016; 34(1): 94–106. Reference Source\n\nEghbalzadeh K, Sabashnikov A, Weber C, et al.: Impact of preoperative elevated serum creatinine on long-term outcome of patients undergoing aortic repair with Stanford A dissection: A retrospective matched pair analysis. Ther Adv Cardiovasc Dis. 2018; 12(11): 289–298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRettig TCD, Peelen LM, Geuzebroek GSC, et al.: Impact of intraoperative hypotension during cardiopulmonary bypass on acute kidney injury after coronary artery bypass grafting. J Cardiothorac Vasc Anesth. 2017; 31(2): 522–528. PubMed Abstract \| Publisher Full Text\n\nde Oliveira Sá MPB, Nogueira JRC, Ferraz PE, et al.: Risk factors for low cardiac output syndrome after coronary artery bypass grafting surgery. Rev Bras Cir Cardiovasc. 2012; 27(2): 217–223. PubMed Abstract\n\nKang J, Yun S, Cho YS, et al.: Post-intensive care unit depression among critical care survivors: A nationwide population-based study. Jpn J Nurs Sci. 2020; 17(2): e12299. PubMed Abstract \| Publisher Full Text\n\nLawson EA, Holsen LM, Desanti R, et al.: Increased hypothalamic-pituitary-adrenal drive is associated with decreased appetite and hypoactivation of food-motivation neurocircuitry in anorexia nervosa. Eur J Endocrinol. 2013; 169(5): 639–647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZakkar M, Guida G, Suleiman MS, et al.: Cardiopulmonary bypass and oxidative stress. Oxid Med Cell Longev. 2015; 2015; 189863. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell JD, Grocott HP, Phillips-Bute B, et al.: Cytokine secretion after cardiac surgery and its relationship to postoperative fever. Cytokine. 2007; 38(1): 37–42. PubMed Abstract \| Publisher Full Text\n\nSchumer EM, Chaney JH, Trivedi JR, et al.: Emergency coronary artery bypass grafting: Indications and outcomes from 2003 through 2013. Tex Heart Inst J. 2016; 43(3): 214–219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPignay-Demaria V, Lespérance F, Demaria RG, et al.: Depression and anxiety and outcomes of coronary artery bypass surgery. Ann Thorac Surg. 2003; 75(1): 314–321. PubMed Abstract \| Publisher Full Text\n\nBootsayatham N, Vanichkul N, Sindhu S, et al.: The relationships among serum creatinine level, ejection fraction, depression, cardio pulmonary bypass time, physical condition and extubation time in patients with elective open heart surgery. Journal of the Royal Thai Army Nurses. 2016; 17(3): 115–122. Reference Source\n\nKyle Simmons W, Burrows K, Avery JA, et al.: Depression-related increases and decreases in appetite: dissociable patterns of aberrant activity in reward and interoceptive neurocircuitry. Am J Psychiatry. 2016; 173(4): 418–428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimmons WK, Burrows K, Avery JA, et al.: Appetite changes reveal depression subgroups with distinct endocrine, metabolic, and immune states. Mol Psychiatry. 2020; 25(7): 1457–1468. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJayakumara S, Borrellib M, Milan Z, et al.: Optimizing pain management protocols following cardiac surgery: A protocol for a national quality improvement study. Int J Surg Protoc. 2019; 14: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOvayolu Ö, Ovayolu N, Aytaç S, et al.: Pain in cancer patients: Pain assessment by patients and family caregivers and problems experienced by caregivers. Support Care Cancer. 2015; 23(7): 1857–1864. PubMed Abstract \| Publisher Full Text\n\nPaulsen Ø, Laird B, Aass N, et al.: The relationship between pro-inflammatory cytokines and pain, appetite and fatigue in patients with advanced cancer. PLoS One. 2017; 12(5): e0177620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang JM, An J: Cytokines, inflammation and pain. Int Anesthesiol Clin. 2007; 45(2): 27–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrunkemeier DM, Cassara JE, Dalton CB, et al.: The narcotic bowel syndrome: clinical features, pathophysiology, and management. Clin Gastroenterol Hepatol. 2007; 5(10): 1126–1139, quiz 1121–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiffen PJ, Derry S, Moore RA: Impact of morphine, fentanyl, oxycodone or codeine on patient consciousness, appetite and thirst when used to treat cancer pain. Cochrane Database Syst Rev. 2014; 2014(5): CD011056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan der Schaaf M, Vermeulen H, Storm-Versloot MN, et al.: Effectiveness of lactulose syrup after cardiac surgery. Appl Nurs Res. 2004; 17(1): 48–54. PubMed Abstract \| Publisher Full Text\n\nLainscak M, Blue L, Clark AL, et al.: Self-care management of heart failure: Practical recommendations from the patient care committee of the heart failure association of the European society of cardiology. Eur J Heart Fail. 2011; 13(2): 115–126. PubMed Abstract \| Publisher Full Text\n\nAndreae C, Strömberg A, Arestedt K: Prevalence and associated factors for decreased appetite among patients with stable heart failure. J Clin Nurs. 2016; 25(11–12): 1703–1712. PubMed Abstract \| Publisher Full Text\n\nPunniyakotti S, Begum B, Kumar A: A case report on digoxin intoxication in a clinical pharmacist's perspective. Asian Journal of Pharmaceutical and Clinical Research. 2018; 11(4): 18. Publisher Full Text\n\nDavis KD, Cheng JC: Differentiating trait pain from state pain: A window into brain mechanisms underlying how we experience and cope with pain. Pain Rep. 2019; 4(4): e735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeal PC, Goes TC, Ferreira da Silva LC, et al.: Trait vs. state anxiety in different threatening situations. Trends Psychiatry Psychother. 2017; 39(3): 147–157. PubMed Abstract \| Publisher Full Text\n\nDietz TM, Lisbeth U: The surgical hospital patient's nutritional risks: A literature review. Klinisk Sygepleje. 2012; 26: 9–22.\n\nMignini EV, Scarpellini E, Rinninella E, et al.: Impact of patients nutritional status on major surgery outcome. Eur Rev Med Pharmacol Sci. 2018; 22(11): 3524–3533. PubMed Abstract \| Publisher Full Text\n\nMakaronidis JM, Neilson S, Cheung W, et al.: Reported appetite, taste and smell changes following Roux-en-Y gastric bypass and sleeve gastrectomy: Effect of gender, type 2 diabetes and relationship to post-operative weight loss. Appetite. 2016; 107: 93–105. PubMed Abstract \| Publisher Full Text\n\nBryant EJ, Malik MS, Whitford-Bartle T, et al.: The effects of bariatric surgery on psychological aspects of eating behaviour and food intake in humans. Appetite. 2020; 150: 104575. PubMed Abstract \| Publisher Full Text\n\nFigshare: PRISMA checklist for ‘Factors related to loss of appetite in postoperative cardiac surgery patients: A systematic review’. http://www.doi.org/10.6084/m9.figshare.14473188" }	[ { "id": "92460", "date": "13 Sep 2021", "name": "Bingyang Ji", "expertise": [ "Reviewer Expertise The blood conservation and organ protection during cardiopulmonay bypass. Improving the prognosis of extracorporeal life support." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 systematically reviewed the factors related to appetite loss after cardiac surgery, which is a quite interesting topic. I have several suggestions:\nThe INTRODUCTION section introduced background knowledge of cardiac surgery. I think the authors should discuss less about it and merge this part with the BACKGROUND section.\n\nI have some concerns on the methods of systematic review. The authors listed PRISM flowchart, some important data were not reported. After excluding duplications, 3246 articles were excluded because they were not published in English/Thai, did not report heart surgery patients or other reasons. The authors should better report the exact number of patients excluded in every step. This may allow replication by other researchers.\n\nTable 2 reported Quality Assessment of the Evidence by GRADE Guideline, and all six articles were of high or moderate quality. I suggest the authors reported more details on assessing the article. It's better to not just report whether it is precise, but report the details of how you decide it's precise.\n\nI recommend the authors to ask for language editing, as some syntax errors existed. e.g. Page 6, Paragraph 4, Line 6, 'Pain is both a rapidly (state variable) and slowly (trait variable) changing factor. '\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "7222", "date": "29 Sep 2021", "name": "Samoraphop Banharak", "role": "Author Response", "response": "The INTRODUCTION section introduced background knowledge of cardiac surgery. I think the authors should discuss less about it and merge this part with the BACKGROUND section. Response: Thank you for suggestion. The introduction and Background to an academic paper are different things and both are essential. The Introduction comes first and is a relatively short section of the manuscript which sets the study we are reporting in its widest context. However, the Background is the place to begin to focus on what, specifically, we investigated. In other words, the INTRODUCTION is the overview, however, BACKGROUND is more scope and focus of what is the phenomenon of this study. For these reasons, we would like to separate and keep these two parts if possible. I have some concerns on the methods of systematic review. The authors listed PRISM flowchart, some important data were not reported. After excluding duplications, 3246 articles were excluded because they were not published in English/Thai, did not report heart surgery patients or other reasons. The authors should better report the exact number of patients excluded in every step. This may allow replication by other researchers. Response: We would like to say thanks for this useful comment and agree with this suggestion. We can add more detail about how many articles were excluded for each reason. There were 347 for not published in English/Thai; 1,245 not heart surgery patients; 109 animal study; and 1,545 not research article. Table 2 reported Quality Assessment of the Evidence by GRADE Guideline, and all six articles were of high or moderate quality. I suggest the authors reported more details on assessing the article. It's better to not just report whether it is precise, but report the details of how you decide it's precise. Response: Thank you so much for useful suggestion. We have followed the GRADE Guideline from Joanna Briggs Institute and provide the standard reported table of GRADE following \"Quality Assessment of the Evidence by GRADE Guideline.\" However, we can add more detail about the reason for giving score for risk of bias, precision, directness, and consistency at the Table 2. Unclear = The researchers did not indicate or reported how they designed for controlling confounding factors and dealing with missing data Precise = The researchers described how to measure their primary and secondary outcomes and used appropriate statistic for their data analysis Direct = The researchers demonstrated they measured primary and secondary outcomes in intervention and control groups in the same way Consistent = The researchers demonstrated they measured primary and secondary outcomes in intervention and control groups in the same construct and measurement I recommend the authors to ask for language editing, as some syntax errors existed. e.g. Page 6, Paragraph 4, Line 6, 'Pain is both a rapidly (state variable) and slowly (trait variable) changing factor.” Response: Thank you so much for suggestion. We have edited this sentence as \"Pain is both a rapid (state variable) and slow (trait variable) changing factor.\" Moreover, this manuscript will be proved again by the English native speaker before publishing the final version." } ] }, { "id": "289513", "date": "26 Jun 2024", "name": "Masato Ogawa", "expertise": [ "Reviewer Expertise 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\nThis manuscript is a well-organized systematic review summarizing loss of appetite in cardiac surgery patients, providing clinically valuable information. Furthermore, loss of appetite, and subsequent malnutrition, are highly likely to affect patient prognosis, making this an extremely important report. The methodology, synthesis, and presentation of results in this systematic review adhere to guidelines and are appropriate.\nI have one point to raise:\nTable 3 summarizes the included studies, but how was loss of appetite, the focus of this review, diagnosed? There is no clinical consensus on how to diagnose loss of appetite, and summarizing the diagnostic criteria used in previous studies would be very helpful. Please add this information to Table 3.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [ { "c_id": "11925", "date": "28 Jun 2024", "name": "Samoraphop Banharak", "role": "Author Response", "response": "Thank you so much for your excellent suggestion. We are also concerned about the diagnosis or criteria to define loss of appetite. We have provided this information in the data extraction table when extracting data, and we can add it to Table 3 by adding one more column. The criteria or diagnosis for loss of appetite in the six included studies were defined by using the symptoms inventory or scale such as the Gastrointestinal Symptom Frequency and Symptom Distress Scale, The Self-Reporting Occurrences of Symptoms, the Cardiac Surgery Symptom Inventory, and the Cardiac Symptoms Survey. These will be added to Table 3 for the revised version." } ] } ]	1	https://f1000research.com/articles/10-350
https://f1000research.com/articles/12-1193/v1	25 Sep 23	{ "type": "Case Report", "title": "Case Report: Hives and faints, an unusual affair", "authors": [ "Saket Toshniwal", "Jiwan Kinkar", "Sunil Kumar", "Sourya Acharya", "Madhukar Tikas", "Isha Sahai", "Benumadhab Ghosh", "Suhit Naseri", "Jiwan Kinkar", "Sunil Kumar", "Sourya Acharya", "Madhukar Tikas", "Isha Sahai", "Benumadhab Ghosh", "Suhit Naseri" ], "abstract": "Cholinergic urticaria (CholU) is a rare condition characterized by itchy hives in the form of 1-4 mm small, raised wheals on skin, which are short-lived for duration of 15 to 20 minutes; they are caused by stimuli associated with sweating such as from physical exercise. CholU is also known as cholinergic angioedema urticaria. Hereby, we present a case report of a 42-year-old male with ChoIU who presented with systemic manifestation in the form of recurrent attacks of syncope. Diagnosis was made after a detailed history taking and all cardiac and neurological evaluations done that were normal, and widespread literature research was done to rule out other causes of syncope as systemic symptoms are rarely seen in ChoIU. His IgE antibodies levels were highly increased. He was managed with nonsedating antihistamines and health education regarding the rare condition.", "keywords": [ "Cholinergic urticaria", "sweating", "nonsedating antihistamics", "syncope" ], "content": "Introduction\n\nCholinergic urticaria (ChoIU) is one of the rare forms of physical urticaria. It is characterized by itchy, pinpoint size wheals which are generally triggered by heat, exercise, mental and physical stress, foul smell and consumption of spicy food. ChoIU is mostly a local reaction and systemic manifestations are rare, seen only in 10% of these cases. Following the exposure to the trigger factor, the onset of CholU occurs rapidly within a few minutes. The average duration of ChoIU symptoms is about 80 minutes, although they can continue up to one to two hours.1–3\n\nThe rarely observed severe cases of CholU, a wheal-flare reaction, may be seen along with systemic involvement, characterized by symptoms like difficult breathing, wheezing, or abdominal pain.4 Sweating associated with urticaria is the main diagnostic feature of CholU in addition to a type I allergic reaction.5 The age group in which CholU is most commonly seen in 20 to 30 years, and there is no sex predominance reported among ChoIU patients.\n\nThe diagnosis is made based on the patient’s medical history and physical examination, although provocation tests are necessary to confirm CholU. The diagnosis of CholU has previously been made using strenuous exercise and passive warming to increase the body’s core temperature.2 Another test involved injecting 100 g of methacholine intradermally with 0.1 mL of saline solution, which resulted in a positive CholU reaction within a minute.2 Recently, two approaches have been proposed to examine patients with CholU suspicions: (i) Patients first exercise for 30 minutes on a treadmill or bicycle trainer, increasing their heart rate by 3 beats per minute; (ii) a passive warming test: a person stays in a 42 degrees Celsius (°C) bath. Following an increase in body temperature of 1°C above the baseline, patients continue the passive warming test for an additional 15 minutes. The presence of tiny wheals during the test and for 10 minutes after it stops indicates a positive result.6\n\nWe share our case to raise awareness of the disorder, shed additional light on the diagnosis, and minimise unnecessary costly studies and delays in diagnosis due to the rarity of systemic involvement seen in CholU.\n\n\nCase presentation\n\nA 42-year-old Asian man labourer by occupation who had previously fainted after physical activity visited the emergency room in our hospital. Before the incidence of fainting, a rash had been previously noticed. The patient also provided a history of syncopal attacks twice after arguments with relatives which begun with severe itch, salivation and blurry vision.\n\nFurther investigation revealed that the patient had previously had papular lesions (Figure 1), itching, and burning after being exposed to various stimuli, including sunshine, hot meals, unpleasant odours, emotions, and exercise, as well as comparable bouts of fainting. The bout of fainting was followed by an unplanned recovery. The wheals and rash also disappeared. There was no history of similar occurrences in either the family or the individual, and there was no family history of any other allergies. After the patient was examined, it was discovered that his cardiac profile, comprising an electrocardiogram, a 2D echocardiogram, a stress test, and holter monitoring, were all within normal limits. Similar to this, the neurological profile, which involved testing including brain MRIs, electroencephalograms, and biochemical analyses, was completed and confirmed to be normal. Provocation tests were done via heavy exercise and passive warming to elicit an allergic response, and were found to be positive for cholinergic urticaria. A section from skin biopsy from anterior chest wall (Figure 2) showed thinned-out and atrophied lining epithelium (panel A and B). Deeper tissue showed increased collagenosis, loss of adnexal structures and scattered chronic lymphocytic inflammatory infiltrate in superficial dermis on histopathology as shown in Figure 2, panel C. Polymorphic light eruption which is seasonal (generally occurs in spring) and has symmetrically distributed polymorphic skin lesions was also ruled out; maculopapular cutaneous mastocytosis was also ruled out as the lesions did not urticate within a few minutes when rubbed; similarly, Sweets Syndrome was also ruled out as the rash did not have a pseudo vesicular aspect. Other differential diagnoses like mast cell activation syndrome, erythema annulare centrifugum, bullous pemphigoid, angioedema, amonf others, were also ruled out based on specific signs and symptoms of each. When all the other causes of syncope were ruled out, serum IgE measure was done which was grossly elevated to 1800 IU/L (Normal range 1.31–165.5 IU/L).\n\nBased on the above history, clinical picture, and investigations, after a thorough literature search, a diagnosis of CholU with cardiovascular involvement as syncope and allergic vasodilation or hyperventilation with stress and exercise was made. The patient was treated with oral fexofenadine 120mg bd, to which he responded, and as of follow-up after six months, he was symptom-free on therapy.\n\n\nDiscussion\n\nCholU is a rare form of inducible urticaria, and was reported by Duke in 1924 for the first time.3,5 In the present case the patient reported with highly pruritic pinpoint wheals associated with the very infrequent systemic symptom of fainting during physical exercise. According to previous reports CholU is generally seen in the age group of 20 to 28 years2; however, in the present case the patient was 42 years old. Based on a previous hypothesis, it is said that patients with CholU are usually hypersensitive to their own stress which results in the development of wheals. According to a theory that has been put forth, patients typically become hypersensitive to unidentified chemicals in their perspiration and develop skin wheals.6,7 The patient in the current report experienced a similar response. The pathogenesis of CholU has been attributed to disturbed AChE. A number of systemic manifestations of CholU have been documented, including dysphagia, dysphonia, inspiratory stridor, lower airway symptoms like wheezing, coughing, chest tightness, and dyspnea, gastrointestinal involvements like nausea, vomiting, abdominal pain, and diarrhoea, and cardiovascular manifestations like presyncope, syncope, and palpitations.6–9 A study by Vadas et al. on 19 cases of ChoIU with cardiovascular system involvement having syncopal attack revealed mast cell degranulation as acteylcholine levels rise.4 According to a Davis et al. study, local pruritus and urticaria are caused by degranulation from skin mast cells, whereas systemic symptoms are thought to be caused by histamine released from circulating basophils.10\n\nIn this case, the exact mechanism of fainting and its association with CholU cannot be postulated. Because systemic involvement in this disorder is rather uncommon, there is frequently a lag between the beginning of symptoms and diagnosis. Additionally, treatment for a normally treatable ailment is delayed and other pointless studies are recommended in between. Non-sedating antihistamines are the mainstay of treatment for CholU, while ketotifen, montelukast, propranolol, and danazol have been suggested in cases where the antihistamines are ineffective.\n\nNon-sedating antihistaminics (nsAH1) are recommended as the first line of treatment for ChoIU and trigger factor escape, under the most recent guidelines of EAACI/GA2LEN/EDF/UNEV9 consensus recommendations for CholU management.\n\n\nConclusions\n\nProper history taking and thorough knowledge about CholU even though it is a rare condition will prove helpful in advising proper investigations as well as avoiding unnecessary investigations. This will ultimately help in saving time, money and mental trauma to the patient. Effective education about CholU and Allergic vasodilation and hyperventilation in presence of mental and physical stressors will aid in understand the triggers and avoiding them; and thus will help in successful management of this unusual and rare condition.\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nSchwartz RA MD, MPH, Chief Editor Elston DM MD: Cholinergic Urticaria Clinical Presentation Dermatology Updated: Jun 06.2022. Reference Source\n\nRujitharanawong C, Tuchinda P, Chularojanamontri L: Nattacha Chanchaemsri, and Kanokvalai KulthananCholinergic Urticaria: Clinical Presentation and Natural History in a Tropical CountryCholinergic Urticaria: Clinical Presentation and Natural History in a Tropical Country. Biomed. Res. Int. 2020 May 24; 2020: 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaggu AK, Rakesh P: Asthma and Immunology Rash before shock: A case of cholinergic urticaria associated with syncope. Srivastava Indian Journal of Allergy. Jan-Jun 2016; 30(1): 42. Publisher Full Text Reference Source\n\nVadas P, Sinilaite A, Chaim M: Cholinergic Urticaria with Anaphylaxis: An Underrecognized Clinical Entity. J Allergy Clin Immunol Pract. 2016 Mar-Apr; 4(2): 284–291. PubMed Abstract \| Publisher Full Text\n\nBito T, Sawada Y, Tokura Y: Pathogenesis of cholinergic urticaria in relation to sweating. Allergol. Int. 2012; 61: 539–544. PubMed Abstract \| Publisher Full Text\n\nAsady A, Ruft J, Ellrich A, et al.: Cholinergic urticaria patients of different age groups have distinct features. Clin. Exp. Allergy. 2017 Dec; 47(12): 1609–1614. Publisher Full Text Reference Source\n\nKim JE, Eun YS, Park YM, et al.: Clinical Characteristics of Cholinergic Urticaria in Korea. Ann. Dermatol. 2014 Apr; 26(2): 189–194. PubMed Abstract \| Publisher Full Text\n\nSeo J-H, Kwon J-W: Epidemiology of urticaria including physical urticaria and angioedema in Korea. Korean J. Intern. Med. 2019 Mar; 34(2): 418–425. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagerl M, Altrichter S, Borzova E, et al.: The definition, diagnostic testing, and management of chronic inducible urticarias - the EAACI/GA2LEN/EDF/UNEV consensus recommendations 2016 update and revision. Allergy. 2016; 71(6): 780–802. PubMed Abstract \| Publisher Full Text\n\nDavis RS, Remigio LK, Schocket AL, et al.: Evaluation of a patient with both aquagenic and cholinergic urticaria. J. Allergy Clin. Immunol. 1981; 68: 479–483. PubMed Abstract \| Publisher Full Text" }	[ { "id": "270954", "date": "17 May 2024", "name": "Ocílio Ribeiro Gonçalves", "expertise": [ "Reviewer Expertise Dermatology", "Neurology and Cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n.Minor points: This is a very interesting case report with a good management description. However, the authors could explain better what is the relationship between vasovagal syncope, acetylcholine, histamine and mast cell and basophils in the discussion. The pathophysiology of Cholinergic urticaria (ChoIU) is essential to understand the clinical presentations of the patient.\n\nMajor points: In discussion it is also necessary that the authors explain why the treatment with an antihistamine (like fexofenadine) could turn the patient symptom-free, based on the pathophysiology of the disease. A simple antihistamine could add quality of life to the patient treating an important cardiovascular symptom presentation.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "284821", "date": "02 Jun 2024", "name": "Thamir A Kubaisi", "expertise": [ "Reviewer Expertise Dermatology and venereology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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-Cholinergic urticaria (CholU) is not a rare problem comprising about 30% of physical urticaria. However the associated syncopal attack is considered a sporadic condition -The signs and symptoms of CholU were aggravated by heavy exercise using a bicycle treadmill or stair-stepping within 15 minutes -According to the modified urticaria activity score, twice-daily form for CholU. The severity depends on\nDuration of skin lesions at attach Number of hives at each check (or number of palms covers pinpoint hives) Itch, tingling intensity\n-The accompanying symptoms such as generalized discomfort, acquired hypo hidrosis, nausea, vomiting and headache are missed in this case report. -Please read this paper carefully: Kubaisi, TA. \"Regular sports exercises as a supportive therapeutic choice in cholinergic urticaria.\" Journal of Pakistan Association of Dermatologists 33.4 (2023): 1298-1305.‏ -Ethical consideration was absent. -There are no clear objectives.: my advice is: the syncopal attach in a young person could be considered as rare presentation of CholU that may be missed by physician.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "11987", "date": "10 Jul 2024", "name": "Saket Toshniwal", "role": "Author Response", "response": "Respected Reviewer, Thank you for reviewing the article and providing us with your expert opinion. The article has been refined and rectified as per your comments. Thank you." } ] } ]	1	https://f1000research.com/articles/12-1193
https://f1000research.com/articles/13-333/v1	23 Apr 24	{ "type": "Study Protocol", "title": "Analysis of the effectiveness and efficiency of the Indonesian metastatic bone disease of unknown origin algorithm (INA-MBD): time to diagnosis and cost to diagnosis: Quasi-experimental study", "authors": [ "Yuni Artha Prabowo Putro", "Teguh Aryandono", "Irianiwati Widodo", "Rahadyan Magetsari", "Dibyo Pramono", "Muhammad Phetrus Johan", "Moh Asri Abidin", "Ardanariswara Wikantyasa", "A Faiz Huwaidi", "Paramita Ayu Saraswati", "Teguh Aryandono", "Irianiwati Widodo", "Rahadyan Magetsari", "Dibyo Pramono", "Muhammad Phetrus Johan", "Moh Asri Abidin", "Ardanariswara Wikantyasa", "A Faiz Huwaidi", "Paramita Ayu Saraswati" ], "abstract": "Background Patients with Metastatic Bone Disease (MBD) often presenting with complaints of pain and multiple osteolytic lesions findings. Remarkably, 30% of these cases exhibit an undetected primary lesion. Hence, categorizing them as MBD of unknown origin. The diagnostic processes of patients with MBD of unknown origin typically takes up to four months, rendering it as a catastrophic disease with the second-highest financial burden. Given its urgency, it is necessary to develop a systematic and evidence-based consensus for managing cases of MBD with an unknown origin.\n\nPurpose This study aimed to enhance the effectiveness and efficiency of treating patients with MBD of unknown origin through the application of the INA-MBD algorithm.\n\nResearch method A quasi-experimental study with a pretest and post-test design was conducted with a total of 128 patients who met the inclusion and exclusion criteria. The patients were consecutively enrolled and categorized into two groups: the intervention group with the INA-MBD algorithm and the non-intervention group without the INA-MBD algorithm. The primary outcomes were the cost and time to diagnose MBD of unknown origin. The proposed measuring tool was the INA-MBD algorithm. Furthermore, for the cost-to-diagnosis variable, an extra measurement tool was used, which were summaries of the patient’s medical bill including hospital stays and medical procedures. The analysis of data related to the time-to-diagnosis variable was conducted using the Log Rank regression test, and cost-to-diagnosis variable was carried out using co-variance test.", "keywords": [ "metastatic bone disease", "Neoplasm", "unknown primary", "algorithm", "management", "cost effectiveness", "diagnosis" ], "content": "Introduction\n\nPatients diagnosed with Metastatic Bone Disease (MBD) of unknown origin are at greater risk of higher mortality and morbidity rates, mainly due to skeletal-related events, with a solely 5% survival rate over a 5-year period.1 In three-quarters of MBD cases, the diagnosis of primary malignancy is made after the patient’s condition deteriorates, typically around four months into the progression of the disease.2 Alarmingly, it has been brought to attention that late establishment of the primary cancer diagnosis significantly contributes to treatment delay up to 86%.3 The prolonged process of diagnosing the unknown origin of MBD is further exacerbated by a series of routine laboratory examinations, including tumor markers.4\n\nPrognosis of patients with MBD of unknown origin highly relies on the timing of diagnosis and treatment. There is no current guideline on the selection of preliminary examinations to identify primary lesions in MBD.1 This lack of consensus adds to the financial burden, with Indonesian national health insurance allocating 18% of the total budget for catastrophic diseases to the care of cancer patients.5 In the United States, the direct cost of MBD is approximately $75,329, double the cost of treating cancer patients without MBD, at $31,382 per year.6,7 Common diagnostic procedures involve a series of laboratory tests such as tumor markers and ALP, X-rays of the chest and affected limbs, CT scans, MRIs, bone scans, and PET scans, followed by bone or organ biopsies. These procedures contribute to prolonged diagnosis times and increased financial burdens, intensifying the complexities associated with addressing MBD-related challenges.\n\nCertainly, we have developed the INA-MBD algorithm based on our literature review and clinical experience in managing patients with MBD of unknown origin. This algorithm provides tailored recommendations for prioritizing supporting examinations based on the patient’s clinical condition. Diverging from conventional methods, the screening checks in the INA-MBD algorithm are not executed sequentially and exhaustively. The main objective is for an early and secure biopsy to speed up the diagnostic process, leading to reduced further examination costs without compromising the success of diagnosing MBD of unknown origin.\n\nObjective: The objective of this study was to assess the impact of implementing the INA-MBD algorithm on the direct treatment costs and the time to diagnose primary malignancy in patients with MBD of unknown origin.\n\n\nProtocol\n\nThis research employed a quasi-experimental pre-test and post-test design, categorizing participants into an intervention group and a non-intervention group. The intervention group utilized the INA-MBD algorithm for diagnosing patients with MBD of unknown origin, while the non-intervention group used the intra-hospital conventional diagnosis algorithm.\n\nIn this study, the intervention group consisted of consecutively collected patients from inpatients, outpatients, and emergency departments diagnosed with MBD of unknown origin until reaching the required sample size. The INA-MBD algorithm was applied for the intervention group. For the non-intervention group, medical record data was utilized from MBD-diagnosed patients with unknown primary lesions between 2018 and 2022 at RSUP Dr. Sardjito (Yogyakarta, Indonesia) and Dr. Wahidin Sudirohusodo (South Sulawesi, Indonesia).\n\n\n\na. Patients with a final diagnosis of MBD\n\nb. Patients without a history of malignancy\n\nc. Patients who agreed to participate in the this study and signed the written informed consent form.\n\n\n\na. Patients with incomplete medical record data\n\nb. Patients who did not undergo operative procedures.\n\nThis multicentre research was conducted at two tertiary referral hospital in Indonesia, RSUP Dr. Sardjito in Yogyakarta and Dr. Wahidin Sudirohusodo in South Sulawesi.\n\nData collection\n\nConsecutive sampling was employed, including all patients meeting the inclusion criteria until the required sample size was met.\n\nIndependent variable\n\nThe INA-MBD algorithm (Figures 1 and 2) will be used as the independent variable. The interventional group will use the INA-MBD algorithm as a management guideline, while the non-interventional group relied on retrospective data from medical records.\n\nThis figure is our original creation synthesized from both literature and our clinical experience.\n\nShow steps and list of management in patients with MBD of unknown origin without pathological fracture. CXR (Chest X-Ray), IHC (Immunohistochemistry), CPC (Clinicopathological conference)/ multidisciplinary team discussion\n\nThis figure is our original creation synthesized from both literature and our clinical experience.\n\nShow steps and list of management in patients with MBD of unknown origin with pathological fracture. CXR (Chest X-Ray), IHC (Immunohistochemistry), CPC (Clinicopathological conference)/multidisciplinary team discussion.\n\nThe dependent variables were time-to-diagnosis and cost-to-diagnosis. Confounding variables, such as gender, age, type of primary cancer, and metastatic location, were derived from medical records for the non-interventional group and other various sources for the interventional group.\n\nBivariate analysis between time to diagnose or cost to diagnose and the use of the INA-MBD algorithm will employ an independent T-test, with Mann-Whitney as an alternative method if the data exhibit abnormal distribution. For the analysis of time to diagnose, the survival rate will be assessed using Kaplan-Meier and log-rank regression tests. To evaluate the hypothesis concerning the cost to diagnose, a covariance test will be employed to analyse the regression mean of both groups.\n\nAfter the completion of the study, we will publish it in an indexed journal or conference.\n\nThe study was in the data collection period and is planned to finish collecting data by December 2024.\n\nThis research will be registered on Researchregistry.com.\n\n\nDiscussion\n\nThe diagnostic challenge in identifying primary malignancies often leads to patients with MBD of unknown origin being misdiagnosed with primary bone tumors or hematological malignancies. An additional complication is that not all MBD patients have a cancer history, with 71% being diagnosed with their primary malignancy only after a clinical deterioration.2 In addition, therapeutic approaches for MBD necessitate concurrent treatment for the primary malignancy.\n\nThe diagnostic process for primary malignancy in MBD typically involves a series of relatively non-invasive imaging examinations. Tsukamoto et al. (2021) advocate for plain X-rays, CT scans, and MRI for all patients with bone lesions. If bone destruction is observed without periosteal reaction, then metastasis is suspected, prompting further examinations like abdominal, thoracic, or pelvic CT scans, tumor markers, serum electrophoresis, Bence Jones protein, and bone scans or PET/CT scans. Biopsy is reserved for the conclusion after completing all imaging and laboratory examinations.8 Tumor marker examinations, including CEA, CA-125, CA19-1, and AFP, exhibit low sensitivity and specificity in determining the primary malignancy in MBD of unknown origin. Tumor markers are not exclusively produced by malignant cells, making serum tumor markers more relevant for therapy monitoring or determining cancer prognosis.4\n\nThe extensive use of radiological examinations extends the time required to establish a primary malignancy diagnosis, contributing to an increased financial burden. Therefore, there is a crucial need for selective use of supportive imaging examinations. Imaging examinations of the gastrointestinal and female reproductive organs often result in waste of time and resources. Thus, not recommended as routine examinations in diagnosing primary lesions in MBD of unknown origin.1 Bone scans alone cannot confirm malignancy origin in MBD and still require a bone survey for lesion confirmation. The cost-effectiveness of a bone survey influences our preference for it over a bone scan.9\n\nMore invasive examinations, such as biopsy, can confirm the origin of the primary malignancy in MBD patients. Biopsy can be performed on metastatic bone lesions, with or without pathological fractures, and on lesions suspected to be primary malignancies. Bone biopsies can confirm the origin of malignancy up to 70%.10\n\nWe have devised an algorithm named INA-MBD through clinical and evidence-based methods. The INA-MBD algorithm offers guidelines for managing patients with MBD of unknown origin, in concordance with the patient’s clinical condition, whether or not pathological fractures are present. This aims to provide more selective guidance in choosing imaging modalities. The INA-MBD algorithm excludes serum tumor marker examinations as a diagnostic tool for the reasons explained above. It also serves as a guide for performing biopsies not only on suspected primary lesions but also on bone lesions. This research seeks to evaluate the effectiveness and efficiency of using the INA-MBD algorithm in diagnosing primary malignancies in patients with MBD of unknown origin. The measurable variables representing the effectiveness of the INA-MBD algorithm include time-to-diagnosis and the cost-effectiveness of managing patients with MBD of unknown origin.\n\nThis study has Ethical approval from The Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada- Dr. Sarjito General Hospital on 09 Jan 2023 and the protocol number is KE/FK/0042/EC/2023 Patients who agreed to participate in the study signed the written consent form.\n\n\nAuthor contributions\n\nConceptualization: (Y.A.P., A.W., T.A., I., R.M., D.P.) Data Curation: (Y.A.P., D.P., A.W., P.A.S., A.F.) Formal Analysis: (Y.A.P.)., A.W., T.A., I., R.M., D.P) Investigation: (Y.A.P., A.W., P.A.S., A.F) Methodology: (Y.A.P., D.P., A.W., M.P.J., M.A.A.) Software: (Y.A.P., P.A.S., A.F.) Supervision: (T.A., I., RM., D.P., M.P.J., M.A.A.) Validation: (Y.A.P., T.A. I., R.M., D.P., M.P.J., M.A.A.) Writing original draft: (Y.A.P., A.W., P.A.S., A.F.) Writing review & editing: Y.A.P., T.A., I., R.M., D.P., M.P.J., M.A.A., A.W., P.A.S., A.F.).", "appendix": "Data availability\n\nNo data are associated with this article.\n\nSpirit Outcome Checklist is available at Zenodo: Checklist for” Analysis of the effectiveness and efficiency of the Indonesian metastatic bone disease of unknown origin algorithm (INA-MBD) time to diagnosis and cost to diagnosis: Quasi Experimental Study”, DOI 10.5281/zenodo.10901730.\n\n\nReferences\n\nTakagi T, Katagiri H, Kim Y, et al.: Skeletal metastasis of unknown primary origin at the initial visit: A retrospective analysis of 286 cases. PLoS One. 2015; 10(6): e0129428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKitagawa Y, Yamaoka T, Yokouchi M, et al.: Diagnostic Value of Plain Radiography for Symptomatic Bone Metastasis at the First Visit. J. Nippon. Med. Sch. 2018; 85(6): 315–321. Publisher Full Text Reference Source\n\nGondhowiardjo S, Hartanto S, Wirawan S, et al.: Treatment delay of cancer patients in Indonesia: A reflection from a national referral hospital. Med. J. Indones. 2021; 30(2): 129–137. Publisher Full Text\n\nPerkins GL, Slater ED, Sanders GK, et al.: Serum Tumor Markers - American Family Physician.2003. Reference Source\n\nBudi E, Juniartha P, Pinem R, et al.: Heru Ganes Santoso.2020.\n\nDiCaprio MR, Murtaza H, Palmer B, et al.: Narrative review of the epidemiology, economic burden, and societal impact of metastatic bone disease. Ann. Jt. 2022; 7: 28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulman KL, Kohles J: Economic burden of metastatic bone disease in the U.S. Cancer. 2007; 109(11): 2334–2342. Publisher Full Text\n\nTsukamoto S, Kido A, Tanaka Y, et al.: Current overview of treatment for metastatic bone disease. Curr. Oncol. 2021; 28(5): 3347–3372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Sullivan GJ: Imaging of bone metastasis: An update. World J. Radiol. 2015; 7(8): 202–211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDatir A, Pechon P, Saifuddin A: Imaging-guided percutaneous biopsy of pathologic fractures: A retrospective analysis of 129 cases. Am. J. Roentgenol. 2009; 193(2): 504–508. PubMed Abstract \| Publisher Full Text" }	[ { "id": "273174", "date": "30 May 2024", "name": "Kevin Christian Tjandra", "expertise": [ "Reviewer Expertise Meta-Analysis", "In-Vivo Research", "Orthopaedic Research", "Oncology Research", "Surgery Research", "Clinical Research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors, Thank you for the opportunity given. This is an interesting in silico research. However, several adjustments are required. To summarize, this research aimed to to enhance the diagnosis of Metastatic Bone Disease (MBD) of unknown origin using the INA-MBD algorithm. Conducted on 128 patients, the quasi-experimental study found that applying the algorithm improved efficiency and reduced costs compared to traditional methods, addressing a high-cost and urgent medical issue. In aim to improving the article, listed below are some concerns that were found so maybe editor can consider doing more review on these matters:\nTitle\n\nThe title has the clarity and conciseness\n\nAbstract\n\nBackground and purpose: the background abstract is well served. Methods: Key improvements include clarifying that patients were randomized to ensure comparability, specifying that the INA-MBD algorithm is the primary diagnostic tool, and detailing how cost analysis was performed. The research also mentioned adjustments for confounding variables and subgroup analyses to increase the robustness of findings. Additionally, reporting baseline characteristics ensured group comparability and notes on follow-up procedures and criteria for evaluating diagnostic accuracy and efficiency were added.\n2.\n\nIntroduction\n\nThe introduction could be improved by incorporating a clearer explanation of the INA-MBD algorithm's development process and evidence supporting its effectiveness. Additionally, providing specific examples or preliminary results demonstrating the algorithm's impact on diagnostic speed and cost reduction would strengthen the introduction. This would help establish the relevance and potential benefits of the algorithm more convincingly.\n3.\n\nMethod To improve the research method, provide a detailed description of the INA-MBD algorithm and how it differs from the conventional diagnosis algorithm, explaining the rationale behind selecting the retrospective data for the control group and ensuring comparability with the intervention group. Include a justification for the sample size calculation, considering the expected effect size and statistical power. Clarify the criteria for patients with a final diagnosis of MBD, specifying diagnostic tests or clinical criteria used, and ensure comprehensive exclusion criteria to avoid confounding variables. Detail how data collection was standardized across different settings and time points, ensuring data quality and completeness, especially for the retrospective control group. Expand on how confounding variables were controlled or adjusted for during the analysis, using multivariate analysis techniques to account for potential confounders. Provide a more detailed plan for the statistical analysis, including assumptions checked for the tests used, handling of missing data, and any sensitivity analyses planned. Include a section on ethical considerations, such as IRB approval and measures to ensure patient confidentiality and data security. Elaborate on the dissemination plan, including specific journals or conferences targeted and any plans for sharing findings with participating hospitals and patients. Provide a detailed timeline of the study phases, including data collection, analysis, and dissemination stages, ensuring alignment with the projected completion date. Confirm the study's registration on Researchregistry.com or provide an update if the registration is complete, including the registration number. 4.\n\nDiscussion\nIn discussing the research, it's crucial to address various aspects for improvement. Firstly, highlighting specific examples or case studies illustrating misdiagnoses of primary bone tumors or hematological malignancies in MBD patients can elucidate the clinical consequences, including treatment delays and prognosis implications. Additionally, elaborating on the evidence-based diagnostic process for primary malignancy in MBD patients, supported by literature, aids in understanding the rationale behind imaging examination sequences and their limitations. Critically analyzing the use of tumor markers in diagnosing primary malignancies provides insights into their sensitivity, specificity, and relevance in clinical practice, supported by relevant studies. Further, detailing the selective use of supportive imaging examinations, with specific criteria for their application, and comparing the diagnostic accuracy, cost-effectiveness, and time efficiency of different modalities, allows for informed decision-making. Discussion on the role of biopsy in confirming primary malignancy origins, including its accuracy, safety, and implications for treatment, should be incorporated, supported by evidence. Introducing the INA-MBD algorithm requires a comprehensive description of its development process, key components, and how it addresses current diagnostic limitations, emphasizing its potential impact in clinical practice. Clear objectives and hypotheses for evaluating the INA-MBD algorithm, along with the methodology for assessing its effectiveness and efficiency, are essential. Additionally, specifying outcome measures, such as time-to-diagnosis and cost-effectiveness, and addressing potential confounding variables in the analysis are crucial. Lastly, discussing the research's implications on clinical practice and healthcare policy, offering recommendations for INA-MBD algorithm implementation, and suggesting areas for future research would provide a comprehensive outlook. 5.\n\nConclusion\nThe conclusion is well-executed\n\n6.\n\nAdditional Information\nThe additional information provides sufficient information\nI hope the suggestions above will improve the article to be indexed. Once again, thank you for the opportunity given.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11739", "date": "09 Jul 2024", "name": "Yuni Artha Prabowo Putro", "role": "Author Response", "response": "Thank you for your kind responses and suggestions. We have adjusted and improved several parts of our manuscript based on the reviewer's comments. Abstract Methods: This research will be conducted using a quasi-experimental design, meaning the sample will receive the intervention without randomization. We chose this design due to the small number of patients with metastatic bone disease of unknown origin, as indicated by both the literature and our clinical experience. Therefore, we will use retrospective data from electronic medical records for the control group, which did not use INA-MBD as a management algorithm, and prospective data for the intervention group, which will use INA-MBD. This decision was made to ensure we have a sufficient sample size. To assess the cost-effectiveness of the INA-MBD algorithm, we only included and analyzed the costs based on ICD.9 coding. This approach was chosen to minimize rate variations between the two hospitals where our research was conducted. The baseline characteristics of the data samples will be presented descriptively. The data includes initials, age, initial diagnosis, details of supporting examinations, and categorization into control or intervention groups. The final diagnosis will be recorded as the primary malignancy if identified, or as MBD of unknown origin if not identified. The diagnosis of primary malignancy or MBD is based on the histopathological result. Introduction The INA-MBD algorithm was developed through a rigorous scientific process. We conducted a systematic review, registered in the research registry with the unique number reviewregistry1457. The results of our review are currently under review in a Scopus-indexed journal. The diagnostic and management patterns of this algorithm were preliminarily tested in our hospital with a limited sample. Both in theory and practice, the INA-MBD algorithm has proven effective in reducing the time required to diagnose primary malignancy in MBD cases and in lowering costs due to fewer supporting examinations. Method Development of INA-MBD Algorithm The INA-MBD algorithm is a management protocol for MBD of unknown origin, developed based on systematic reviews and our clinical experience treating MBD patients at a tertiary referral hospital. INA-MBD differs from other MBD management algorithms because it tailors subsequent examinations based on whether the patient has experienced a pathological fracture. Additionally, we excluded tumor marker testing from the INA-MBD algorithm due to the low level of evidence supporting their use in diagnosing primary malignancies in MBD cases. Our algorithm incorporates a clinicopathological conference (CPC) as a diagnostic tool. The widely used algorithm by Tsukamoto et al. (Curr. Oncol. 2021, 28(5), 3347-3372; https://doi.org/10.3390/curroncol28050290) significantly differs from the INA-MBD algorithm we propose. Rationale to use retrospective data We used retrospective data for the control group due to the limited number of patients with MBD of unknown origin at our center. Therefore, we employed retrospective data for the control group (where the INA-MBD algorithm had not been applied) and prospective data for the intervention group (where the INA-MBD algorithm was applied). Sample size To determine the sample size, we used the formula for the difference in continuous data between the two groups. With a 95% confidence interval and 80% power, and based on the study by Kitagawa et al. (2018), the time required to diagnose MBD of unknown origin was 16 weeks. The researchers considered a meaningful difference to be 8 weeks. Thus, the minimum sample size needed was 63 per group or 126 in total. Final diagnosis of MBD definition In diagnosing cases of MBD, we used radiological examinations to identify bone lesions where the primary tumor was unknown and there was no previous history of malignancy at presentation. The primary malignancy of MBD was confirmed using the histopathological examination. Data collection Patients diagnosed with MBD of unknown origin based on clinical and radiological examinations will be consecutively collected. Those meeting the inclusion and exclusion criteria will be included in the sample. Data will be collected using a standardized case report form (Supplementary Material) for each sample and both intervention and control groups. Patients with incomplete medical records will be excluded. IRB approval This study has been approved by the Medical and Health Research Ethics Committee (MHREC) of the Faculty of Medicine, Public Health, and Nursing at Universitas Gadjah Mada – Dr. Sardjito General Hospital, with reference number KE/FK/0162/EC/2024. Dissemination plan Upon completion of this study, the findings will be published in a Scopus-indexed journal with a Q2-Q1 quartile ranking. Additionally, the results will be presented at the Continuing Orthopaedic Education meeting held by the Indonesian Orthopaedic Association We have revised our latest manuscript to incorporate the comments and suggestions from the reviewers." } ] } ]	1	https://f1000research.com/articles/13-333
https://f1000research.com/articles/13-1/v1	03 Jan 24	{ "type": "Data Note", "title": "A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence", "authors": [ "Riham Ayoubi", "Maryam Fotouhi", "Charles Alende", "Kathleen Southern", "Carl Laflamme", "NeuroSGC/YCharOS/EDDU collaborative group", "ABIF consortium", "Riham Ayoubi", "Maryam Fotouhi", "Charles Alende", "Kathleen Southern" ], "abstract": "Protein phosphatase 2A is a serine/threonine phosphatase with activity dependent on an associated regulatory subunit, serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D). PPP2R5D is the δ isoform in the B56 family of regulatory subunits. Abundantly expressed in the brain and involved in a broad range of cellular processes, PPP2R5D plays an essential role in modulating key neuronal pathways and signalling. Pathogenic mutations in the PPP2R5D gene are linked to clinical symptoms characterized by neurodevelopmental delay, intellectual disability, and autism spectrum disorders. The etiology of these genetic disorders remains unknown, which can partly be due to the lack of independently characterized antibodies. Here we have characterized six PPP2R5D commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.", "keywords": [ "Uniprot ID Q14738", "PPP2R5D", "Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform", "antibody characterization", "antibody validation", "Western Blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nProtein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates various cellular functions including cell metabolism, cell cycle, DNA replication, transcription and translation, signal transduction, cell mobility and apoptosis.1–4 PP2A has a heterotrimeric form consisting of 3 subunits: catalytic, scaffold and regulatory.5 The specificity and activity of the PP2A enzyme is regulated by the associated regulatory subunit,which controls substrate selectivity and activity of PP2A.6–9\n\nThe PP2A-B56 family of the regulatory subunit has 5 isoforms; α, β, γ, δ, and ε. The PPP2R5D gene encodes serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D).9,10 This δ isoform is highly expressed in neuronal tissues and is crucial for cell cycle regulation. It controls the ability of PP2A to negatively regulate PI3K/AKT signalling pathways and modulates glycogen synthase kinase-3 β and cyclin-dependent kinase 5 activities by controlling tau phosphorylation.8,11 Missense mutations in PPP2R5D have been linked to neurodevelopmental disorders such as neurodevelopmental delay,12 intellectual disability13–15 and autism spectrum disorder.16–20 The majority of these mutations lead to Jordan’s syndrome, an autosomal dominant illness associated with intellectual disabilities and autism spectrum disorders.20 Exploring the intricate network involving PPP2R5D as it impacts cell cycle regulation in neuronal processes could significantly enhance the potential for uncovering novel treatments for neurodevelopmental disorders. Further research is required and would be facilitated with the availability of high-quality antibodies.\n\nThis research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data.21–23 Here, we evaluated the performance of six commercially-available antibodies for PPP2R5D in Western Blot, immunoprecipitation and immunofluorescence using a knockout based approach. This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of PPP2R5D properties and function.\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.24,25 The first step is to identify a cell line(s) that expresses sufficient endogenous levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the PPP2R5D transcript at RNA levels above the average range of cancer cells analyzed. Parental and PPP2R5D KO HAP1 cells were obtained from Horizon Discovery (Table 1).\n\nFor Western Blot experiments, we resolved proteins from WT and PPP2R5D KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).\n\nA) Lysates of HAP1 WT and PPP2R5D KO were prepared in RIPA buffer, and 30 μg of protein were processed for Western Blot with the indicated PPP2R5D antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 5687, MA5-18066 and MA5-26647, which were titrated to the corresponding dilutions found below, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: ab188323* at 1/10 000, ab188325** at 1/10 000, 5687* at 1/200, GTX106401 at 1/500, MA5-18066* at 1/200, MA5-26647* at 1/200. B) The observed molecular weight of PPP2R5D varies according to the lysis buffer used. HAP1 WT and PPP2R5D KO lysates were prepared in RIPA or IP lysis buffer and the molecular weight of PPP2R5D was assessed by WB. The composition of lysis buffers is indicated in the Methods section. PPP2R5D ran primarily at ~65 kDa or ~75 kDa when lysates were prepared with RIPA or IP lysis buffer, respectively. ab188323 was used at 1/10 000 for Western Blot.\n\nAs per our standard procedure, we next used the antibodies to immunoprecipite PPP2R5D from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the PPP2R5D protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).\n\nHAP1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated PPP2R5D antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for Western Blot with the indicated PPP2R5D antibody. For Western Blot, ab188323 was used at 1/5000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. Monoclonal antibody, Recombinant antibody.\n\nFor immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure. First, HAP1 WT and PPP2R5D KO cell lines were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the six PPP2R5D antibodies were evaluated. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested. The images presented in Figure 3 are representative of the results of this analysis.\n\nHAP1 WT and PPP2R5D KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated PPP2R5D antibodies (first panel) and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI (second panel). Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT cells were outlined with green dashed lines while KO cells were outlined with magenta dashed lines. When the concentration was not indicated by the supplier, which was the case for antibodies ab188325, 5687, GTX106401, MA5-18066* and MA5-26647, we tested antibodies at the concentrations found below. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab188323* at 1/200, ab188325** at 1/300, 5687* at 1/70, GTX106401 at 1/1000, MA5-18066* at 1/1000, MA5-26647* at 1/1000. Bars = 10 μm. Monoclonal antibody, Recombinant antibody.\n\nIn summary, we have screened six PPP2R5D commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect PPP2R5D under our standardized experimental conditions were identified. In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. However, the authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret the antibody characterization data in this study are available on the YCharOS Gateway.26\n\nThe underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.27,28\n\n\nMethods\n\nAll PPP2R5D antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.29 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).\n\n Monoclonal antibody.\n\n** Recombinant antibody.\n\nBoth HAP1 WT and PPP2R5D KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.30 Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat. number 450201).\n\nHAP1 WT and PPP2R5D KO were collected RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat. number 89901) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.\n\nWestern Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were developed with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) Or Clarity Western ECL Substrate from Bio-Rad (cat. number 1705061) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).\n\nAntibody-bead conjugates were prepared by adding 2 μg of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.\n\nHAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western Blot on a precast midi 4-20% Tris-Glycine polyacrylamide gels. VeriBlot for IP Detection Reagent:HRP (Abcam, cat. number ab131366) was used as a secondary detection system at a concentration of 0.1 μg/mL.\n\nHAP1 WT and PPP2R5D KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. WT and KO cells were then plated in a 96-well plate with optically clear flat-bottom as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10 ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL). Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095), 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary PPP2R5D antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.\n\nImages were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water objective lens and scientific CMOS camera (16-bit, 1.97 mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.31 Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for PPP2R5D, https://doi.org/10.5281/zenodo.10108248. 27\n\nZenodo: Dataset for the PPP2R5D antibody screening study, https://doi.org/10.5281/zenodo.10119578. 28\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.\n\nABIF consortium: Claire M. Brown and Joel Ryan.\n\nThank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.\n\nAn earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.10108248).\n\n\nReferences\n\nTung HY, Alemany S, Cohen P: The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle. Eur. J. Biochem. 1985; 148(2): 253–263. Publisher Full Text\n\nAlberts AS, Thorburn AM, Shenolikar S, et al.: Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc. Natl. Acad. Sci. U. S. A. 1993; 90(2): 388–392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlenn GM, Eckhart W: Mutation of a cysteine residue in polyomavirus middle T antigen abolishes interactions with protein phosphatase 2A, pp60c-src, and phosphatidylinositol-3 kinase, activation of c-fos expression, and cellular transformation. J. Virol. 1993; 67(4): 1945–1952. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRonne H, Carlberg M, Hu GZ, et al.: Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol. Cell Biol. 1991; 11(10): 4876–4884. PubMed Abstract\n\nKamibayashi C, Estes R, Lickteig RL, et al.: Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. J. Biol. Chem. 1994; 269(31): 20139–20148. PubMed Abstract \| Publisher Full Text\n\nJanssens V, Longin S, Goris J: PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem. Sci. 2008; 33(3): 113–121. PubMed Abstract \| Publisher Full Text\n\nEichhorn PJ, Creyghton MP, Bernards R: Protein phosphatase 2A regulatory subunits and cancer. Biochim. Biophys. Acta. 2009; 1795(1): 1–15. PubMed Abstract \| Publisher Full Text\n\nLouis JV, Martens E, Borghgraef P, et al.: Mice lacking phosphatase PP2A subunit PR61/B’delta (Ppp2r5d) develop spatially restricted tauopathy by deregulation of CDK5 and GSK3beta. Proc. Natl. Acad. Sci. U. S. A. 2011; 108(17): 6957–6962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCright B, Brothman AR, Virshup DM: Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and b56epsilon (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12. Genomics. 1996; 36(1): 168–170. PubMed Abstract \| Publisher Full Text\n\nJanssens V, Goris J: Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 2001; 353(Pt 3): 417–439. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShang L, Henderson LB, Cho MT, et al.: De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism. Neurogenetics. 2016; 17(1): 43–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiswas D, Cary W, Nolta JA: PPP2R5D-Related Intellectual Disability and Neurodevelopmental Delay: A Review of the Current Understanding of the Genetics and Biochemical Basis of the Disorder. Int. J. Mol. Sci. 2020; 21(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Ligt J , Willemsen MH, van Bon BW , et al.: Diagnostic exome sequencing in persons with severe intellectual disability. N. Engl. J. Med. 2012; 367(20): 1921–1929. Publisher Full Text\n\nGilissen C, Hehir-Kwa JY, Thung DT, et al.: Genome sequencing identifies major causes of severe intellectual disability. Nature. 2014; 511(7509): 344–347. Publisher Full Text\n\nStessman HA, Xiong B, Coe BP, et al.: Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases. Nat. Genet. 2017; 49(4): 515–526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbrahams BS, Arking DE, Campbell DB, et al.: SFARI Gene 2.0: a community-driven knowledgebase for the autism spectrum disorders (ASDs). Mol. Autism. 2013; 4(1): 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasu SN, Kollu R, Banerjee-Basu S: AutDB: a gene reference resource for autism research. Nucleic Acids Res. 2009; 37(Database issue): D832–D836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIossifov I, O’Roak BJ, Sanders SJ, et al.: The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014; 515(7526): 216–221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSatterstrom FK, Kosmicki JA, Wang J, et al.: Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism. Cell. 2020; 180(3): 568–84.e23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSandal P, Jong CJ, Merrill RA, et al.: Protein phosphatase 2A - structure, function and role in neurodevelopmental disorders. J. Cell Sci. 2021; 134(13). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, Ryan J, Biddle MS, et al.Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. Elife. 2023; 12. Publisher Full Text\n\nCarter AJ, Kraemer O, Zwick M, et al.: Target 2035: probing the human proteome. Drug Discov. Today. 2019; 24(11): 2111–2115. PubMed Abstract \| Publisher Full Text\n\nLicciardello MP, Workman P: The era of high-quality chemical probes. RSC Med. Chem. 2022; 13(12): 1446–1459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nBiddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12(12): 1344. Publisher Full Text\n\nAyoubi R, Fotouhi M, Alende C, et al.: Antibody Characterization Report for PPP2R5D.2023. Publisher Full Text\n\nSouthern K: Dataset for PPP2R5D antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18(1): 100–106. PubMed Abstract \| Publisher Full Text" }	[ { "id": "269488", "date": "20 May 2024", "name": "Nadia Bouhamdani", "expertise": [ "Reviewer Expertise Cancer research", "proteomics", "functional genomics", "rare disease research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors characterize six commercially available antibodies for PPP2R5D using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. The main objective of this study is to provide a published report to guide researchers in the field in selecting the most appropriate available antibodies for their specific research needs.\nIn general, this data note article is clear and straight forward; however, important details are lacking. Here are some comments/questions that need to be addressed:\n\nIn the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log2(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available? An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)? I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results). In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11965", "date": "09 Jul 2024", "name": "Kathleen Southern", "role": "Author Response", "response": "Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns. In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log2(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation. Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log2 (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1). 2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity. 3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section. Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section. 4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available? Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences. 5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)? Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research. 6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results). Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results. 7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested. Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B. 8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing. Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1). Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded." } ] } ]	1	https://f1000research.com/articles/13-1
https://f1000research.com/articles/13-782/v1	09 Jul 24	{ "type": "Research Article", "title": "The comparison study on accuracy of peak expiratory flow rate measurement by PRAAN, spirometry, and the mini-Wright peak flow meter", "authors": [ "Sirawich Chaiparnich", "Orapan Poachanukoon", "Natcha Manasilp", "Narongkorn Saiphoklang", "Charturong Tantibundhit", "Pasitpon Vatcharavongvan", "Pattapol Kunumpol", "Phongpan Plienphanich", "Apiwat Pugongchai", "Kanyada Leelasittikul", "Nantavat Prompoom", "Sirawich Chaiparnich", "Natcha Manasilp", "Narongkorn Saiphoklang", "Charturong Tantibundhit", "Pasitpon Vatcharavongvan", "Pattapol Kunumpol", "Phongpan Plienphanich", "Apiwat Pugongchai", "Kanyada Leelasittikul", "Nantavat Prompoom" ], "abstract": "Background PRAAN, a digital peak flow meter, was developed to measure peak expiratory flow rate (PEFR) for asthma monitoring. This study aimed to compare PRAAN’s accuracy to that of spirometry and the mini-Wright peak flow meter for PEFR measurement.\n\nMethods Two cross-sectional studies were conducted in 106 healthy adult participants. Study A (n=56) compared PRAAN with spirometry, while study B (n=50) compared PRAAN with the mini-Wright peak flow meter. PEFR values were collected using crossover studies. Pearson’s correlation and Bland-Altman plots were used to report the relationship and agreement between two measurements, respectively.\n\nResults In study A, 51.8% were female, mean age was 24.3±5.6 years, and PEFR was 480.3±86.8 L/min. PEFR measured by PRAAN had very strongly positive correlation with spirometry (r=0.980, P<0.001). Bland-Altman analysis showed that there was good agreement between them, with a low mean difference of -7.07 liters/minute (95% CI: -40.58 to 26.44 liters/minute) and 92.9%, which were within the limit of agreement (LOA). In study B, 54.0% were female, mean age was 23.5±2.4 years, and PEFR was 495.1±82.7 L/min. PEFR measured by PRAAN and by the mini-Wright peak flow meter showed very strongly positive correlation (r=0.971, P<0.001). Bland-Altman comparison of PEFR between these two measurements showed that there was a very good agreement between them, with a low mean difference of 0.84 liters/minute (95% CI: -38.68 to 40.38 liters/minute) and 94.0%, which were within LOA.\n\nConclusions The accuracy of PRAAN is in the agreement with spirometry and the mini-Wright peak flow meter. The clinical application of PRAAN may potentially lead to a monitoring strategy that healthcare providers can use to improve the management of asthma.", "keywords": [ "agreement", "asthma", "Bland-Altman plots", "correlation", "digital peak flow meter", "peak expiratory flow rate" ], "content": "Introduction\n\nAsthma is a chronic respiratory disease which is characterized by airway inflammation, hyperresponsiveness, and variable expiratory airflow limitation.1 It is increasingly prevalent,2,3 probably because of factors including air pollution,4–6 sedentary lifestyles,7,8 and genetic predisposition.9,10 Diagnosis of asthma is based on clinical characteristics and lung function assessment such as spirometry with bronchodilator testing.1,11,12 However, spirometry results may vary in the same patients and should be carried out by well-trained technicians. In clinical practice, peak expiratory flow rate (PEFR) is measured by peak flow meter to assess airflow limitation of asthmatic patients. It is an indispensable parameter for monitoring airway diseases, particularly asthma,13 and it is recommended as an alternative tool when spirometry is not available.14 A frequent application of PEFR is in home monitoring of asthmatic patients because it is easy to do, and it is inexpensive. In Thailand, patients use mechanical mini-Wright peak flow meters and record their PEFR values in peak flow diaries. For physicians, limited accessibility of spirometry and peak flow meters, poor compliance with manual data collection, and data fabrication are longstanding problems.15\n\nPRAAN (Precise Reliable Advanced Affordable and Novel), under copyright license number 2201002185 is a digital peak expiratory flow device (D-PEF device) designed to function like a peak flow meter. It has direct electronic power, data storage, and built-in analytical functions, and a complementary mobile application serves as an interface for the device. PRAAN is equipped with light interruption sensors and a detachable blower to measure PEFR. Acknowledging the potential advantages offered by PRAAN, a study was conducted to compare its accuracy to that of the gold standard spirometry and the mini-Wright peak expiratory flow meter. A demonstration of substantial agreement between the results would endorse acceptance from the medical field and promote the broader adoption of PRAAN as a reliable alternative.\n\nThis study aimed to compare measurements of PEFR by PRAAN digital peak flow device with measurements by spirometry and by the mini-Wright peak flow meter.\n\n\nMethods\n\nTwo sets of cross-sectional studies were conducted at Thammasat University Hospital, Thailand between December 2022 and June 2023. Study A (n=56) was a comparison of PEFR measured by PRAAN and spirometry, while study B (n=50) was a comparison of PEFR measured by PRAAN and the mini-Wright peak flow meter. In both studies, a cross-over method was done, in which participants in each study were divided into two groups. The first group underwent PEFR measurements using PRAAN, followed by measurements using spirometry or the mini-Wright peak flow meter while the second group underwent the measurements in the opposite order (Figure 1).\n\nHeathy subjects aged 18 years or older were included. Subjects with active respiratory infection (e.g., common cold, COVID-19), inability to perform PEFR measurement or spirometry, current or former smoking, or respiratory diseases including asthma, chronic obstructive pulmonary disease, or any obstructive airway disease were excluded.\n\nEthical approval was obtained from the Human Research Ethics Committee of Thammasat University (Medicine), Thailand (IRB No. MTU-EC-PE-0-154/65, COA No. 254/2022) and the date of approval Decmber 16, 2022. in full compliance with international guidelines such as the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines, and the International Conference on Harmonisation-Good Clinical Practice (ICH-GCP). All methods etwere performed in accordance with these guidelines and regulations. All participants provided written informed consent.\n\nPRAAN is an ergonomic and portable digital peak flow meter meticulously crafted for precise the measurement of PEFR (Figure 2). This device features an internal sensor arrangement utilizing light interruptions for accurate PEFR evaluation (Figure 3). It is comprised of two central elements: a main chassis housing the sensor, display, and controller board, and a detachable blower equipped with perpendicular blades and a mouthpiece holder. The blower’s distinct design enables full immersion, adhering to stringent hospital-grade cleanliness criteria. PRAAN was engineered to endure impacts, chemicals, and thermal fluctuations, and it is made of antibacterial plastic. It can be operated in two distinct modes: standalone mode and mobile application mode, offering real-time PEFR measurements. Additionally, PRAAN considers PEFR factors such as sex, age, and height. A light indicator system of red, yellow, and green lights, signifies lung function levels.\n\nParticipants performed the test by forcefully exhaling through the mouthpiece, with results promptly displayed on the device screen or mobile application. The displayed light indication offered insights into lung function status. PRAAN mandated three vigorous exhalations with a variance of less than 10% between blows to finalize assessment.\n\nParticipants performed this procedure by exhaling forcefully into a specialized device known as a spirometer. Before testing, participants received instructions and the spirometer was meticulously calibrated to ensure accuracy, allowing the spirometer to record volumetric and flow rate data. Pulmonary function parameters including forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC ratio, and PEFR were recorded. Spirometry was performed according to the American Thoracic Society and European Respiratory Society guidelines16–18 using PC spirometer (Vyntus® SPIRO, Vyaire Medical, Inc., Mettawa, IL, USA).\n\nThe mini-Wright peak flow meter (Clement Clarke International Ltd., UK) is a compact handheld device used for assessment of PEFR in our study. This device facilitated precise measurements of exhaled airflow vigor.19 Participants were instructed to exhale forcefully through the mouthpiece, prompting the movable marker to traverse the calibrated scale and indicated the highest achieved flow rate. The test was performed with three attempts, with one-minute breaks between attempts. The maximal value of the three efforts was recorded for the final analysis.\n\nData are presented as number (%) and mean ± standard deviation (SD). Pearson’s correlation was used to determine the relationship of PEFR measured by PRAAN and spirometry or the mini-Wright peak flow meter. Bland-Altman plots were used to determine agreement of PEFR obtained by PRAAN and spirometry or the mini-Wright peak flow meter. In the Bland-Altman plot, the mean difference between the two methods of measurement was calculated and compared among the subjects to make sure that the difference lies within the limit of agreement between the two methods. The 95% limits of agreement, which represent where 95% of the future differences between the two methods would be, was calculated via the use of standard deviation. The proportional bias also calculated by utilizing a linear relationship between the difference for each paired measurement. Two-tailed P-values of less than 0.05 were considered statistically significant. All data analyses were done on SPSS version 26.0 software https://www.ibm.com/products/spss-statistics (IBM Corp., Armonk, NY, USA).\n\n\nResults\n\nData were collected from 56 subjects in study A (51% females, mean age 24.3 years) and 50 subjects in study B (54% females, mean age 23.5 years). PEFR measured by PRAAN was 473.2±85.6 L/minute and 495.9±76.1 L/minute in studies A and B, respectively. PEFR measured by spirometry and the mini-Wright peak flow meter were 480.3±86.8 L/minute and 495.1±82.7 L/minute in studies A and B, respectively (Table 1).\n\nThere were very strongly positive correlations between PEFR measured by PRAAN and by spirometry (r=0.980, P<0.001), as well as PEFR measured by PRAAN and by the mini-Wright peak flow meter (r=0.971, P<0.001) (Table 2).\n\nIn study A, the comparison between PRAAN and spirometry revealed no statistically significant difference in PEFR values (P>0.05). The comparison of PEFR between PRAAN and spirometry by Bland–Altman analysis (as shown in Figure 4) showed that there was a good agreement between them, with a low mean difference of -7.07 liters/minute (95% CI: -40.58 to 26.44 liters/minute) and only 4 of 56 measurements (7.1%) were outliers (Figure 4). No proportional bias was detected (B=-0.015, P=0.582).\n\nStudy B compared PEFR between PRAAN and the mini-Wright peak flow meter. The results indicated no statistically significant difference in the PEFR values obtained from the two devices (P>0.05). The Bland–Altman comparison of PEFR between PRAAN and the mini-Wright peak flow meter (as shown in Figure 5) showed that there was a very good agreement between them, with a low mean difference of 0.84 liters/minute (95% CI: -38.68 to 40.38 liters/minute) and only 3 of 50 measurements (6%) were outliers (Figure 5). However, a proportional bias was detected (B=-0.084, P=0.020).\n\nData presented as n (%) or mean±SD\n\nL=liters, m=meters, PEFR=peak expiratory flow rate\n\n\nDiscussion\n\nThis study is a comparison of PEFR measurement by an innovative digital peak flow meter named PRAAN with measurement by the mini-Wright peak flow meter or by spirometry in healthy adult subjects. Our results showed that the accuracy of PRAAN was similar to the accuracy of spirometry and to the accuracy of the mini-Wright peak flow meter.\n\nPRAAN emerges as a portable and economically viable D-PEF device. It’s light indicator system, comprising red, yellow, and green lights, signifies lung function levels or asthma action plans. This design serves to enhance individuals’ awareness of their lung function status, promoting respiratory health awareness, particularly in Thailand’s healthcare setting.\n\nThrough seamless coordination with a companion mobile application, the D-PEF device streamlines daily PEFR collection, enhancing efficiency. The mobile application not only simplifies PEFR measurement but also safeguards against data fabrication, ensuring reliable data acquisition. This integration exemplifies a contemporary approach to PEFR assessment, combining accurate measurement methods with user-friendly technology to enhance respiratory health management.\n\nSpirometry is a foundational technique in pulmonary function assessment.20 It provides valuable insights into lung function and holds widespread clinical utility for diagnosing and monitoring respiratory conditions, aiding in disease identification, severity assessment, and treatment progress evaluation.21,22 However, potential limitations of spirometry are participant effort,23 operator dependency and interpretation difficulty,21,22 and equipment calibration for standardizing protocols to ensure accurate results.24 In the context of our study, spirometry served as a reference method, facilitating the comprehensive evaluation of PEFR.\n\nPEFR is recommended as a crucial parameter for asthma management according to the international guidelines.1 The mini-Wright peak flow meter is simple to operate, cheap and convenient to assess airflow limitation and variability.25,26 However, it is noteworthy that variations in technique and effort among subjects may influence to PEFR results.15,27 Our study design is similar to a study comparing 11 peak flow meters on the market by Folgering H, et al.27 A parallel performance of PRAAN and the mini-Wright peak flow meter in PEFR assessment is evident through the lack of statistically significant differences in our study. The robust positive correlation and substantial agreement emphasize their concordance, strengthening PRAAN’s credibility as a measurement tool. These outcomes suggest that PRAAN can serve as a suitable alternative to the mini-Wright device, offering a convenient and reliable option for healthcare professionals in assessing PEFR. Our results showed that the accuracy of PRANN digital peak flow meter is equivalent to the gold standard spirometry and the mini-Write peak flow meter.\n\nPRAAN’s performance is on par with established methods, and its user-friendly design and potential economic benefits reinforce its relevance in respiratory health management. Moreover, the adherence to PEFR monitoring is limited, which is why it is not widely used in clinical practice. A study by Miles JF, et al showed that using electronic media in PEF-based self-management plans may help to improve asthma outcomes.28 Our digital peak flow meter could increase compliance by using the mobile application.\n\nThere are a few limitations in our study. Firstly, the sample size was small, however study results could detect the correlation of two measurements. Secondly, the study population was not generalized distribution due to narrow age ranges. However, we focused on a healthy population in this study. A large study is needed to verify PRAAN accuracy for all age groups of normal subjects and asthmatic patients.\n\n\nConclusions\n\nPRAAN digital peak flow meter is an accurate instrument for assessing PEFR, in agreement with spirometry and the mini-Wright peak flow meter. It demonstrates reliability and potential for wider application with addition to clinical practice, managing respiratory health, including asthma.\n\n\nEthics and consent\n\nEthic approval was obtained from the Human Research Ethics Committee of Thammasat University (Medicine), Thailand (IRB No. MTU-EC-PE-0-154/65, COA No. 254/2022) and the date of approval December 16 2022, in full compliance with international guidelines such as the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines, and the International Conference on Harmonisation-Good Clinical Practice (ICH-GCP). All methods were performed in accordance with these guidelines and regulations. All participants provided written informed consent.\n\n\nClinical trial registration\n\nthaiclinicaltrials.org with number TCTR20230816002", "appendix": "Data availability\n\nFigshare: The comparison study on accuracy of peak expiratory flow rate measurement by PRAAN, spirometry, and the mini-Wright peak flow meter. DOI: https://doi.org/10.6084/m9.figshare.24511357.v1\n\nPlease ensure your data availability statement adheres to the following format:\n\n• Study_PEF.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to thank Michael Jan Everts, Faculty of Medicine, Thammasat University, for proofreading this manuscript. The location for conducting out the research was provided by Thammasat University Research Unit in Allergy and Respiratory Medicine, and Medical Diagnostics Unit, Thammasat University Hospital, Thailand.\n\n\nReferences\n\nGlobal Initiative for Asthma: Global strategy for asthma management and prevention 2023.Reference Source\n\nWong QYA, Lim JJ, Ng JY, et al.: An updated prevalence of asthma, its phenotypes, and the identification of the potential asthma risk factors among young Chinese adults recruited in Singapore. World Allergy Organ J. 2023; 16(3): 100757. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDharmage SC, Perret JL, Custovic A: Epidemiology of asthma in children and adults. Front. Pediatr. 2019; 7: 246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuarnieri M, Balmes JR: Outdoor air pollution and asthma. Lancet. 2014; 383(9928): 1581–1592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChatkin J, Correa L, Santos U: External environmental pollution as a risk factor for asthma. Clin. Rev. Allergy Immunol. 2022; 62(1): 72–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTiotiu AI, Novakova P, Nedeva D, et al.: Impact of air pollution on asthma outcomes. Int. J. Environ. Res. Public Health. 2020; 17(17). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoodley I, Williams L, Thompson C, et al.: Evidence for lifestyle interventions in asthma. Breathe (Sheff.). 2019; 15(2): e50–e61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu KD, Forno E, Radom-Aizik S, et al.: Low fitness and increased sedentary time are associated with worse asthma-The National Youth Fitness Survey. Pediatr. Pulmonol. 2020; 55(5): 1116–1123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ndi Palmo E , Cantarelli E, Catelli A, et al.: The predictive role of biomarkers and genetics in childhood asthma exacerbations. Int. J. Mol. Sci. 2021; 22(9). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBijanzadeh M, Mahesh PA, Ramachandra NB: An understanding of the genetic basis of asthma. Indian J. Med. Res. 2011; 134(2): 149–161. PubMed Abstract\n\nAyuk AC, Uwaezuoke SN, Ndukwu CI, et al.: Spirometry in asthma care: A review of the trends and challenges in pediatric practice. Clin. Med. Insights Pediatr. 2017; 11: 117955651772067. Publisher Full Text\n\nRoychowdhury P, Badwal J, Alkhatib F, et al.: Spirometry utilization among patients with asthma. J. Asthma Allergy. 2020; 13: 193–203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSitalakshmi R, Poornima KN, Karthick N: The peak expiratory flow rate (PEFR): the effect of stress in a geriatric population of Chennai- A pilot study. J. Clin. Diagn. Res. 2013; 7(2): 409–410.\n\nKirenga BJ, Schwartz JI, de Jong C , et al.: Guidance on the diagnosis and management of asthma among adults in resource limited settings. Afr. Health Sci. 2015; 15(4): 1189–1199. PubMed Abstract \| Publisher Full Text\n\nRedline S, Wright EC, Kattan M, et al.: Short-term compliance with peak flow monitoring: results from a study of inner city children with asthma. Pediatr. Pulmonol. 1996; 21(4): 203–210. PubMed Abstract \| Publisher Full Text\n\nMiller MR, Crapo R, Hankinson J, et al.: General considerations for lung function testing. Eur. Respir. J. 2005; 26(1): 153–161. Publisher Full Text\n\nMiller MR, Hankinson J, Brusasco V, et al.: Standardisation of spirometry. Eur. Respir. J. 2005; 26(2): 319–338. Publisher Full Text\n\nGraham BL, Steenbruggen I, Miller MR, et al.: Standardization of spirometry 2019 update. An official American Thoracic Society and European Respiratory Society technical statement. Am. J. Respir. Crit. Care Med. 2019; 200(8): e70–e88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeVrieze BW, Modi P, Giwa AO: Peak flow rate measurement. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023.\n\nHeijkenskjöld Rentzhog C, Janson C, Berglund L, et al.: Overall and peripheral lung function assessment by spirometry and forced oscillation technique in relation to asthma diagnosis and control. Clin. Exp. Allergy. 2017; 47(12): 1546–1554. PubMed Abstract \| Publisher Full Text\n\nSchermer TR, Robberts B, Crockett AJ, et al.: Should the diagnosis of COPD be based on a single spirometry test? NPJ Prim. Care Respir. Med. 2016; 26: 16059. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPierce R: Spirometry: an essential clinical measurement. Aust. Fam. Physician. 2005; 34(7): 535–539. PubMed Abstract\n\nBorg BM, Hartley MF, Fisher MT, et al.: Spirometry training does not guarantee valid results. Respir. Care. 2010; 55(6): 689–694. PubMed Abstract\n\nCoenraad Frederik N, Koegelenberg FS: Elvis Malcolm Irusen: Guideline for office spirometry in adults, 2012. S. Afr. Med. J. 2013; 103: 103. Publisher Full Text\n\nPerks WH, Tams IP, Thompson DA, et al.: An evaluation of the mini-Wright peak flow meter. Thorax. 1979; 34(1): 79–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDekker FW, Schrier AC, Sterk PJ, et al.: Validity of peak expiratory flow measurement in assessing reversibility of airflow obstruction. Thorax. 1992; 47(3): 162–166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFolgering H, Brink W v, Heeswijk C, et al.: Eleven peak flow meters: a clinical evaluation. Eur. Respir. J. 1998; 11(1): 188–193. PubMed Abstract \| Publisher Full Text\n\nMiles JF, Tunnicliffe W, Cayton RM, et al.: Potential effects of correction of inaccuracies of the mini-Wright peak expiratory flow meter on the use of an asthma self-management plan. Thorax. 1996; 51(4): 403–406. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "311272", "date": "27 Sep 2024", "name": "Yasuo To", "expertise": [ "Reviewer Expertise Asthma and Allergy" ], "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\nTo find out the feasibility of PEFR meter for patients with respiratory diseases, it would be essential to test it with both healthy volunteers and individuals with various respiratory diseases. Otherwise, it is not possible to identify patients and to evaluate disease severity. The range of PEFR value is broad if you enroll both healthy volunteers and patients. Analysis using only healthy volunteers would not prove the machine’s potential for patients.\nIs there any influence to the machine by temperature, humidity, meteorological changes, or altitude? As far as the machine is precise one, the authors must describe how these circumstances affect to ability of PEFR-measurement when using this machine.\nIs this machine needs AC-power? If so, it would not be able to use some of the developing countries with a lack of power. The authors should clarify not only merit but demerit of this machine.\nMini-lite PEFR meter is used worldwide. If the authors think this machine may be one of the alternatives of Mini-lite, Detailed comparison with Mini-lite must be shown not only good part but also bad part.\nLimitations of the device should be shown in discussion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-782
https://f1000research.com/articles/12-1104/v1	04 Sep 23	{ "type": "Research Article", "title": "Non-tuberculous mycobacterium isolations from tuberculosis presumptive cases at the National Tuberculosis Reference Laboratory Kenya, 2018–2019", "authors": [ "George Kamau", "Zakayo Mwangi", "Joel Bargul", "Maurice owiny", "Nellie Mukiri", "Immaculate Kathure", "Beatrice Khamala", "Nassoro Mwanyalu", "Richard Kiplimo", "Raphael Lihana", "Zakayo Mwangi", "Joel Bargul", "Maurice owiny", "Nellie Mukiri", "Immaculate Kathure", "Beatrice Khamala", "Nassoro Mwanyalu", "Richard Kiplimo", "Raphael Lihana" ], "abstract": "Background: Mycobacterial pathogens are among the top causes of diseases in humans. In Kenya, incidences of Non-Tuberculous Mycobacteria (NTM) species have steadily been on the increase. Most NTMs are resistant to first line treatment of tuberculosis and have a challenge in timely and accurate diagnosis. Misdiagnosis has led to prescribing anti-tuberculosis regimens to patients suffering from NTM. We aimed to determine the most prevalent Non-Tuberculous Mycobacterium in Kenya. Methods: We reviewed records from the National Tuberculosis Reference Laboratory (NTRL) Laboratory information management system (LIMS) between January 2018 and December 2019 for the patients on surveillance. All isolates were cultured in Mycobacterial Growth Indicator Tubes (MGIT) and incubated for detection using BACTEC™ MGIT™ system. Those with negative acid-fast bacilli (AFB) growth and negative for Mycobacterium Tuberculosis Complex Species (MTBC) protein-MPT64 were suggestive of NTM infections, which were sub-cultured in MGIT and characterized using Line Probe Assay (LPA) GenoType® MTBDR CM/AS. Descriptive and bivariate analysis was done. Results: Of the total 24,549 records reviewed, 167(0.7%) were NTM isolates. Males comprised of 74.2% (124/167), and the mean age was 42 years (SD±16), age group 35-44 years had the highest NTM at 26.3% (44/167). Nairobi had 12.6% (21/167), Mombasa 10.8% (18/167), Kilifi and Meru each had 7.8% (13/167). Eleven isolated species comprised of Mycobacterium intracellulare 35.3% (65/167), M. fortuitum at 27% (48/167), and M. scrofulaceum at 10.2% (17/167). Previously treated patients had higher NTM [63.5% (106/167)] than Drug-resistant follow-up patients [26.9% (45/167)]. Coinfection with HIV was at 27.5% (46/167). Conclusion: Previously treated patients should have an additional screening of NTMS, and drug susceptibility testing should be done before initiation of treatment.", "keywords": [ "Nontuberculous Mycobacteria", "Kenya", "Mycobacterium avium Complex", "Mycobacterium tuberculosis", "Prevalence" ], "content": "Introduction\n\nHumans are often affected by Mycobacterium infections. In developing countries, non-tuberculous mycobacterial (NTM) infections have been overshadowed by high incidences of tuberculosis. This trend is, however, fast-changing. The number of new infections is increasing due to the growing number of immune-deficient patients (Ferreira et al., 2002) and nonsterile endoscopic medical devices (Duarte et al., 2009). Pulmonary diseases caused by NTM have been on the increase worldwide (Hoefsloot et al., 2013). In 2014 estimated prevalence of pulmonary tuberculosis caused by NTM was estimated to be 33–65 per 100,000 persons (Morimoto et al., 2014).\n\nGlobally, positive acid-fast bacilli (AFB) specimens are assumed to develop Mycobacterium tuberculosis (MTBC) and are therefore managed by anti-tuberculosis agents whereas some are put under speculated areas (Mertaniasih et al., 2017). The high prevalence of AFB leads to isolation of mycobacterial illness, meaning that more patients with AFB-positive samples have been administered unsuitable and, more so, unnecessary empirical anti-tuberculosis treatment (Ngayo et al., 2015). As a result of this incorrect treatment and high treatment letdown, the mortality of patients with lung infections is increasing. Therefore, appropriate diagnosis and regular monitoring are crucial in monitoring and treating patients (Aksamit et al., 2014).\n\nIn Kenya, data regarding the burden of pulmonary illness is scanty, partly due to constraints in surveillance and diagnosis of mycobacterial species. Distribution of these species (NTM) contributes to the difficulties in interpreting positive culture results. Furthermore, it is not mandatory to report cases of disease to the database at the Ministry of Health; thus, data regarding the epidemiology and distribution of pulmonary disease’s causation is inadequate in Kenya (Jacqueline et al., 2015). The decision to initiate treatment should be influenced by the severity of disease, the risk of progressive NTM-pulmonary disease, the presence of comorbidity and the goals of treatment. Decision to treat remains individualized with consultations and individuals may require a period of longitudinal assessment (symptoms, radiological change and mycobacterial culture results) to inform NTM treatment decisions. To determine the clinical relevance of NTM positive cultures, it is essential to distinguish transient or persistent colonization (which is usually not treated) from true infection (Daley et al., 2020). It’s against this that the current study was undertaken to characterize the NTM\n\n\nMethods\n\nThis study was approved by Kenyatta National Hospital-University of Nairobi Ethics Review Committee (Ref:KNH-ERC/A/306) on September 14, 2020. Waiver for individual informed consent was granted as the study utilized remnant clinical samples and the research posed no greater than minimal risk to the study subjects.\n\nSputum samples were collected in a leak proof container with a volume of >3ml, which were labelled with client name, date of sample collection and client registration number. The samples were accompanied by a duly filled request form and transported in triple package which were received at the National Tuberculosis Reference Laboratory (NTRL)] underwent mycobacterial culture and identification procedures as described in NTRL standard operating procedures (SOP). Where sputum samples were decontaminated and digested using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure. Inoculation into mycobacteria growth indicator tube (MGIT) and Lowenstein-Jensen (LJ) media was done and incubated at 37°C. Growth monitoring for up to 6 and 8 weeks respectively, was done. At the same time, sputum smears were prepared, air dried, heat-fixed then fluorochrome stained with Auramine O. Appearance of mycobacteria as bright yellow fluorescent rods was definitive of correct diagnosis when viewed under a light-emitting diodes microscope. Upon thorough mixing, the tubes were centrifuged at 14,000g for five minutes. The supernatant was gathered in distinct 1.5ml tubes for onward processing. Culture growth in liquid and solid media went through MTB identification using Ag MPT64 assay (capilia), the positive ones were excluded in the study and the negative samples underwent ZN microscopy and presence of AFB indicating a possible NTM.\n\nMycobacterial DNA was extracted from 500 μL of re-suspended colonies using GenoLyse (Hain Lifescience, Nehren,Germany) (106477), according to the manufacturer’s instructions. Briefly, 100ul of lysis buffer (A-LYS) was added to each cryovial containing the resuspended colonies and incubated for five minutes at 95°C after which 100ul neutralization buffer (A-NB) was added and centrifugation was done at 5000g for 10 minutes. The supernatant was transferred to a newly labeled cryovial awaiting PCR. A conventional PCR targeting the hsp65 gene was conducted using the GoTaq Green Master Mix (Promega, Madison, Wisconsin, USA) in a final reaction volume of 13 μl comprising 6.25 μl of 2X GoTaq Hot Start Green Master Mix, 2.5 μl DNA template, 0.25 μl of each of both F-(5′-ACCAACGATGGTGTGTCCAT-3′) and R-(5′-CTTGTCGAACCGCATACCCT-3′) primers at a final concentration of 10 pmoles, and 3.75 μl of nuclease-free water to make up the reaction volume. Thermal cycling conditions were 1 cycle of 94°C for 4 min, 35 cycles of 94°C for 1 min, 57°C for 1 min, 72°C for 1 min and a final extension for 10 min at 72°C. Amplified products were confirmed on a 1% Agarose gel stained with 4.6 μl SYBR safe DNA stain (Invitrogen, Carlsbad, California, USA), and bands at 441 bp were observed in an Ultra Violet gel viewer. The PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were 37°c for 15 min followed by a second incubation at 80°c for 15 min and a final cooling step at 4°C for 5 min.\n\n\nResults\n\nA total 24,549 records were reviewed. Of these, 167 (0.7%) NTMs were isolated (Kamau, 2023). The highest isolation was among individual aged 35-44 years, (46/167, 27.5%). Males comprised 74.2% (124). The mean age (SD) was 42(16) years. The NTM were isolated in 65% (31/47) of the counties; Most samples came from Nairobi County at 12.6% (21/167), then Mombasa 10.8% (18/167), and Kilifi and Meru counties at 7.8% (13/167). HIV positivity rate of 27.5%(46/167). Most NTM were isolated from previously treated patients 63.5%(106/167), drug-resistant follow-up patients 26.9% (45/167) and New patients 9.6%(16/167) [Table 1]. Coinfection with NTM and HIV was at 27.5% (46/167).\n\nA total of eleven NTM species were identified, with frequent ones being Mycobacterium intracellulare at 35.3% (59/167), M. fortuitum at 26.3% (44/167), and M. scrofulaceum at 10.2% (17/167). Majority of NTM had a Smear outcome of No AFB seen [59.3%(99)], Smear 1+ [11.4%(19)], and smear 2+ [10.2%(17)] [Table 2].\n\n\nDiscussion\n\nOur study found that the prevalence of NTM was low AT [0.7%(167/24,549)] and most of the cases were males which was similar to study done by (Fatima & Nm, 2019) on prevalence of Nontuberculous Mycobacterial infection in Non-HIV subjects. The MAC complex is considered to be the most prevalent globally accounting for 86% (Nishiuchi et al., 2017). These findings are consistent with our study that isolated MAC species M.intracellulare [35.3 % (59/167), M.fortuitum 26.3% (44/167) M. scrofulaceum 10.2% (17/167), M.kansasii 9.6% (16/167)] [Table 2]. MAC species are frequently isolated in different environmental sources like water and soil and there is frequent contact with this sources thus increasing its infection to human (van Ingen et al., 2009). Similar findings from other countries have described isolation of MAC species (Namkoong et al., 2016; Nishiuchi et al., 2017). Similar finding found out that M. intracellulare was most prevalent (Epola Dibamba Ndanga et al., 2022). However, MAC was not reported as predominant in other settings i.e. South Pacifica where M.fortuitum complex fewer cases were isolated; in India M.fortuitum was predominant and MAC was not detected. The high infectivity rate of MAC species may be linked to their apparent abundance and distribution in many environmental sources such as water and soil, which increases the ease of dissemination and infection to people. MAC has been strongly linked to NTM Pulmonary Disease (NTMPD); nevertheless, it is unknown how its infectivity connects to NTMPD (Mwangi et al., 2021, 2022).\n\nIn our findings persons within the age group (35-44 years) were most affected. More males were affected suggesting that women could be having a protective effect due to the hormonal variation which triggers a response and clears the bacterial infection (Namkoong et al., 2016; Ji et al., 2020).\n\nPulmonary NTM were mostly found in patients who were previously treated for TB Most of the cases were HIV negative which was inconsistent with what has been reported previously (Stepanyan et al., 2019). In people with HIV/AIDS, NTM infections can occur in various parts of the body, including the lungs, lymph nodes, and skin. These infections can be severe and difficult to treat, especially if the immune system is severely compromised.\n\nMost NTM were isolated from of counties, leading county being Nairobi County with [12.6 %(21/167)], followed by Mombasa County and Kilifi and Meru County. These findings may be inconclusive on factors influencing the geographical distribution of NTM in the different geographical landscapes in Kenya, a more comprehensive study capturing both human and environmental particulars is required for a better understanding of environmental NTM species distribution. Differences in specific NTM species predominance in various environments may in part influence the frequency of pulmonary NTM disease in each geographical location.\n\nThe presence of NTM among the presumptive cases poses a public health challenge and which potentially complicates TB diagnosis and management. The Division of National TB, Leprosy and Lung Diseases Program (DNTLD-P) should put NTM in planning the prevention and management of tuberculosis control, these findings are similar to a study done in Gabon (Epola Dibamba Ndanga et al., 2022).\n\n\nConclusion\n\nMAC was the most prevalent followed by M.fortuitum and M.scrofulaceum. Previously treated patients should have an additional screening of NTMS, and drug susceptibility testing should be done before initiation of treatment, additional screening of NTM’s in should be done in males especially withiinthe age group of (35-44) years.\n\nThe study lacked detailed patient history which hindered identification of clinically relevant NTM and association with NTM-Pulmonary disease. Despite the limitations the study was able to characterize the diversity of NTM", "appendix": "Data availability\n\nfigshare: Non-Tuberculous Mycobacterium isolations from Tuberculosis presumptive cases at the National Tuberculosis Reference Laboratory Kenya, 2018 –2019. https://doi.org/10.6084/m9.figshare.23498426.v1 (Kamau, 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors wish to express their gratitude to all participants who took part in this study, grateful to National Public Health Laboratories- Kenya for granting the access to NTRL and conduct the research and appreciate the assistance accorded by the staffs in their technical support in preparation and analysis of samples in this study.\n\n\nReferences\n\nAksamit TR, Philley JV, Griffith DE: Nontuberculous mycobacterial (NTM) lung disease: The top ten essentials. Respir. Med. 2014; 108(3): 417–425. PubMed Abstract \| Publisher Full Text\n\nDaley CL, Iaccarino JM, Lange C, et al.: Treatment of Nontuberculous Mycobacterial Pulmonary Disease: An Official ATS/ERS/ESCMID/IDSA Clinical Practice Guideline. Clin. Infect. Dis. 2020; 71(4): e1–e36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuarte RSRS, Lourenço MCSMCS, Fonseca LdSLdS, et al.: Epidemic of Postsurgical Infections Caused by Mycobacterium massiliense. J. Clin. Microbiol. 2009; 47(7): 2149–2155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEpola Dibamba Ndanga M, Achimi Agbo Abdul JBP, Edoa JR, et al.: Non-tuberculous mycobacteria isolation from presumptive tuberculosis patients in Lambaréné, Gabon. Tropical Med. Int. Health. 2022; 27(4): 438–444. PubMed Abstract \| Publisher Full Text\n\nFatima and Nm—2019—Prevalence of nontuberculous Mycobacterial infecti.pdf: n.d. Retrieved May 16, 2023. Reference Source\n\nFatima S, Nm A: Prevalence of nontuberculous Mycobacterial infection in Non-HIV subjects of clinically presumed tuberculosis at a tertiary care center, Hyderabad. IP International Journal of Medical Microbiology and Tropical Diseases. 2019; 5(2): 116–122. Publisher Full Text\n\nFerreira RMC, Saad MHF, da Silva MG , et al.: Non-tuberculous mycobacteria I: One year clinical isolates identification in Tertiary Hospital Aids Reference Center, Rio de Janeiro, Brazil, in pre highly active antiretroviral therapy era. Mem. Inst. Oswaldo Cruz. 2002; 97: 725–729. PubMed Abstract \| Publisher Full Text\n\nHoefsloot W, van Ingen J , Andrejak C, et al.: The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: An NTM-NET collaborative study. Eur. Respir. J. 2013; 42(6): 1604–1613. PubMed Abstract \| Publisher Full Text\n\nIntegrated Guideline: n.d.. 467.\n\nJacqueline L, Christine B, Ngayo MO, et al.: Infection rates and correlates of non-tuberculous mycobacteria among tuberculosis retreatment cases in Kenya. Prime Journal of Social Science (PJSS). 2015; 1128–1134.\n\nJi S, Xu W, Sun J, et al.: Retrospective analysis of patients with non-tuberculous mycobacteria from a primary hospital in Southeast China. Sci. Rep. 2020; 10(1): 1060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamau G: Non-Tuberculous Mycobacterium isolations from Tuberculosis presumptive cases at the National Tuberculosis Reference Laboratory Kenya, 2018 –2019. [Dataset]. figshare. 2023. Publisher Full Text\n\nMertaniasih NM, Kusumaningrum D, Koendhori EB, et al.: Nontuberculous mycobacterial species and Mycobacterium tuberculosis complex coinfection in patients with pulmonary tuberculosis in Dr. Soetomo Hospital, Surabaya, Indonesia. Int. J. Mycobacteriol. 2017; 6(1): 9–13. PubMed Abstract \| Publisher Full Text\n\nMorimoto K, Iwai K, Uchimura K, et al.: A Steady Increase in Nontuberculous Mycobacteriosis Mortality and Estimated Prevalence in Japan. Ann. Am. Thorac. Soc. 2014; 11(1): 1–8. PubMed Abstract \| Publisher Full Text\n\nMwangi ZM, Mukiri N, Onyambu FG, et al.: Genetic Diversity of Non-Tuberculous Mycobacteria among Symptomatic TB negative Patients in Kenya. bioRxiv. 2021; p. 2021.10.28.465104. Publisher Full Text\n\nMwangi ZM, Mukiri N, Onyambu FG, et al.: Genetic Diversity of Nontuberculous Mycobacteria among Symptomatic Tuberculosis Negative Patients in Kenya. Int. J. Mycobacteriol. 2022; 11(1): 60. Publisher Full Text\n\nNamkoong H, Kurashima A, Morimoto K, et al.: Epidemiology of Pulmonary Nontuberculous Mycobacterial Disease, Japan. Emerg. Infect. Dis. 2016; 22(6): 1116–1117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNgayo M, Ngayo M, Mutua D: Infection rates and correlates of Non-Tuberculous Mycobacteria among Tuberculosis retreatment cases In Kenya. Prime J. Soc. Sci. 2015; Vol. 4(7): pp. 1128–1134. 2315-5051.\n\nNishiuchi Y, Iwamoto T, Maruyama F: Infection Sources of a Common Non-tuberculous Mycobacterial Pathogen, Mycobacterium avium Complex. Front. Med. 2017; 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStepanyan IE, Zaytseva AS, Bagdasaryan TR, et al.: Nontuberculous mycobacterial co-infection in HIV-negative patients with pulmonary tuberculosis. Eur. Respir. J. 2019; 54(suppl 63). Publisher Full Text\n\nvan Ingen J , Boeree MJ, Dekhuijzen PNR, et al.: Environmental sources of rapid growing nontuberculous mycobacteria causing disease in humans. Clin. Microbiol. Infect. 2009; 15(10): 888–893. PubMed Abstract \| Publisher Full Text" }	[ { "id": "246327", "date": "20 Mar 2024", "name": "Durga Shankar Meena", "expertise": [ "Reviewer Expertise Infectious Diseases" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. In 'abstract' section, patient with negative AFB and MPT 64 ag were analysed? What about AFB +, gene Xpert negative patients? Many of such patients might belonged to NTM group.\n2. Line \"Nairobi had 12.6% (21/167), Mombasa 10.8% (18/167), Kilifi and Meru each had 7.8% (13/167).\" Please complete the sentence.\n3. Conclusion of the abstract is not corroborative with objectives of study. It should be revised.\n4. 'Introduction' last line - it seems unclear.\n5. Methodology - Authors should clearly mention what were the inclusion and exclusion criteria for this study?\n6. Results - Number of patients with HIV positivity and HIV coinfection both sentences are the same.\n7. MAC was commonest species isolated. What were the possible factors for this? It should also be compared with other geographical/national prevalence.\n\n8. What was the proportion of MAC in HIV patients in this study? How many patients had previous tuberculosis? Any data on clinical profile of these patients? Mortality ? Outcomes?\n\n9. Please mention the proportion of rapid growers and slow grower NTM in this study.\nThe major limitation of this study is lack of clinical data correlating to NTM isolation. Methodology is also unclear, how were these samples were selected for NTM isolation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11968", "date": "09 Jul 2024", "name": "George Kamau", "role": "Author Response", "response": "I addressed all the reviews and recommendations made and sent a revised version" } ] }, { "id": "276826", "date": "06 Jun 2024", "name": "Abiona Oluwadamilola Odeyemi", "expertise": [ "Reviewer Expertise Lung infection and lung function in people with chronic illnesses." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 was a good research. It was technically sound and exhibited originality. It was also well structured and it gave adequate detail as to how the organisms were isolated. I would have attached the PDF file of the manuscript with my comments so that my comments on some aspects of the manuscript can be seen here. 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/12-1104
https://f1000research.com/articles/12-1272/v1	05 Oct 23	{ "type": "Clinical Practice Article", "title": "Treatment of acute sub-massive pulmonary embolism with balloon angioplasty and thrombolysis simultaneously", "authors": [ "Mark Christopher Arokiaraj" ], "abstract": "Background: Acute sub-massive pulmonary embolism is a common clinical condition, and it is associated with high mortality and morbidity. This condition is commonly associated with various co-morbidities and clinical circumstances.\nMethods: This is a case report series of 4 patients, wherein the thrombolysis and balloon angioplasty was performed simultaneously using a Cordis 6F diagnostic catheter and later exchanged with a 6F guide catheter in the respective pulmonary artery when a larger balloon was required.\nResults: All these four patients achieved successful revascularization of the respective pulmonary artery. One patient expired 30 hours after the procedure with a significant reduction in the symptoms suddenly, which was likely a second episode of pulmonary embolism. No bleeding manifestations were observed in any of the patients. The other three patients are on follow-up.\nConclusion: Simultaneous coronary balloon dilatation and thrombolysis is a useful method in the treatment of sub-massive acute pulmonary embolism. If needed, higher caliber balloons can be used for the same technique using guide catheters.", "keywords": [ "Acute submissive pulmonary embolism", "Balloon angioplasty", "Simultaneous thrombolysis", "Maceration" ], "content": "Introduction\n\nAcute pulmonary embolism is a common medical condition, and the treatment is often challenging. The incidence of acute pulmonary embolism is in the range of 0.6/1000/year.1 The estimated incidence of deep vein thrombosis is 1-2/1000 persons every year.2 It is the 3rd leading cause of cardiovascular mortality. The burden of deep vein thrombosis and related pulmonary embolism are high.3 These also pose a large economic burden for the health care providers irrespective of the system worldwide.3 The mortality in patients with acute pulmonary embolism is significant, and the exact burden is high when quantified. At present robust data about this condition is not available and the magnitude of the problem is an underestimate. Common challenges are encountered in treating this condition is associated severe breathlessness, co-morbidities and the patient is often moribund. Associated comorbidities or predisposing conditions preclude the treatment methods due to the associated risk of bleeding and mortality. Hence, controversies exist in choosing the method of treatment of this condition, and the best treatment methods at present are catheter directed thrombolysis and low dose thrombolysis. Among the costs involved, the catheter directed thrombolysis is associated with the lowest cost.4 This is by considering the risk of bleeding vs. the benefits achieved, which is primarily the mortality. With the advent of the COVID-19 pandemic, the incidence of venous thrombosis has increased significantly, especially in severe cases of COVID-19.5 At present catheter directed thrombolysis has the best results, and even this is associated with 6.5% cardiac arrest and about 5% hemoptysis.6\n\nCatheter directed thrombolysis is associated with least mortality6 though there are some studies which show definite conclusions regarding the treatment methods is not possible.7 Some studies show similar results with all methods of treatment modalities,8 though many studies claim the benefits9–11 and safety12 of catheter directed thrombolysis. Prompt treatment of this condition is always advised, and treatment delays are associated with increased mortality.13 The current report is descriptive about four male patients encountered with acute sub-massive/severe pulmonary embolism, who were treated with balloon angioplasty/maceration and thrombolysis.\n\n\nMethods\n\nInformed written consent was taken from patients for all procedures. Institutional ethical committee approval has been obtained from Pondicherry Institute of Medical Sciences (PIMS) Institutional ethical review committee, Puducherry. The consent to publish the individual clinical details described in this report was obtained from each patient.\n\nThe patient was a 45-year male who developed acute onset of breathlessness and presented to the emergency ward with acute breathlessness and chest pain. Angiogram was performed, and the coronary angiogram was normal. As the patient had persistent tachycardia, the echocardiogram performed showed dilated right atrium, ventricle, and pulmonary artery. Pulmonary angiogram, which showed dilated right atrium and right ventricle and near total occlusion of the left pulmonary artery (Figure 1, panels A). The patient underwent balloon dilatation, and thrombolysis was performed simultaneously (Figure 1, panels B to F). Accuforce balloon, 4.5mm, was used for balloon dilatation through Cordis 6F diagnostic catheter, and thrombolysis was performed with tenecteplase. 35 mg of Tenecteplase was used with boluses given by hand injections simultaneously with balloon dilatations. There was an immediate improvement in breathlessness, reduction in tachycardia, and the oxygen saturation also improved to 99 percent when the procedure was completed in the cardiac catheterization lab. Flow in the left pulmonary artery improved (Figure 1, panels G and H). The patient had improvement in the general condition, and he was discharged on day three. The right atrium and right ventricular dilatation were also reduced. Deep veins were normal by Doppler evaluation, and hence inferior vena-cava filter was not placed in this patient. This patient is under follow-up for one year, and at present on rivaroxaban and currently asymptomatic.\n\nPanel A shows clots in left pulmonary artery. Panels B to F shows balloon dilatation and thrombolysis simultaneously in left pulmonary artery (panels B to F). Results with clearing of the clots and improvement in oxygen saturation (Panels G and H), 4.5 mm Accuforce balloon was used.\n\nThe 81-year male patient developed breathlessness and chest pain, which was sudden in onset. The patient recently underwent a hip operation for an intertrochanteric fracture on the right side, and recently he was ambulant. The pulse rate at presentation was 140/min, and the patient had severe breathlessness, and by echocardiography, the right atrium and right ventricle were dilated. A pulmonary angiogram showed near total occlusion of the right pulmonary artery (Figure 2A, panel A). The patient underwent thrombolysis with balloon dilatation in the right pulmonary artery, where the clots were visualized (Figure 2A, panels B to E). A 5 mm quantum apex balloon was used initially, and it was improvised with a 6 mm Sterling balloon (Figure 2, panel F). Tenecteplase 35 mg was used in the procedure and was given as boluses after dilution with saline. At the end of the procedure, the clot volume was reduced, and pulmonary artery blood flow improved (Figure 2A, panels G and H). The patient’s symptoms improved, and tachycardia mildly reduced immediately after the procedure. Subcutaneous low molecular weight heparin was given with good results. On day two, the tachycardia and breathlessness were reduced by about 60%. A repeat pulmonary angiogram performed on the third day showed significant clearance of the clots in the right pulmonary artery (Figure 2B). The patient is on regular follow-up for one year and 1 m.\n\nA 59-year male presented with breathlessness and orthopnea for ten days, and this patient had mild pericardial effusion and corpulmonale and severe pulmonary artery hypertension. The patient also had crepitations and collapse of the entire right lung. Angiogram showed a large clot in the right pulmonary artery (Figure 3, panels A and B). Initially, 035 Terumo wire was inserted, and using a peripheral - 5×40 mm balloon, angioplasty was performed in the right pulmonary artery. Thereafter, 014 wire was inserted (Figure 3, panel E), and balloon dilatations using coronary balloons, 2×10 mm, and 4.5×8 mm, were performed (Figure 3, panel F). The patient underwent serial balloon dilatations and simultaneous thrombolysis with streptokinase by hand injection. Partial recanalization of the right pulmonary artery was achieved (Figure 3, panels G to I). Investigations for deep vein thrombosis and any associated malignancies did not reveal any findings. A pulmonary angiogram was repeated after two days, and balloon dilatation with heparin was given during the second intervention for residual thrombi in the right pulmonary artery. The patients’ general condition and breathlessness improved, and he was discharged. One month follow-up showed mild lung expansion compared to the previous results on the right side, and he had mild breathlessness on exertion with pulmonary artery systolic pressure of 45 mmHg.\n\nThis was a 47-year male who presented with breathlessness and cough for one day and had a history of occupation-related long travels. Echocardiography revealed a corpulmonale and right ventricular dysfunction. An inferior vena-cava angiogram showed clots in the left iliac artery (Figure 4, panel A). The patient had large clots in the left pulmonary artery with near-total occlusion (Figure 4, panels B and C). Using Cordis 6F diagnostic catheter, serial balloon dilatations were performed to clots in the left pulmonary artery (Figure 4, panels D and E), and its mid and lower branches using a 5 mm Quantum apex balloon and thrombolysis was performed simultaneously using streptokinase in the left pulmonary artery. A significant improvement in the blood flow of the left pulmonary artery was observed (Figure 4, panels G and H). The patient had relief in breathlessness and chest tightness immediately after the procedure and a significant reduction in oxygen requirement. He was started on low molecular weight heparin and clopidogrel 75mg once a day. For 24 hours post-procedure patient had substantial clinical improvement. 1.3M units of streptokinase were used by dilution in saline and were administered as hand injections. On day two, the patient, being asymptomatic with minimal oxygen support, developed sudden onset of breathlessness and rapid deterioration and death subsequently. The patient possibly could have had an episode of another pulmonary embolism from the deep veins or from the primary source, which was only clinical speculation. No bleeding manifestations were observed before deterioration.\n\n\nDiscussion\n\nIn all the above cases, balloon angioplasty/maceration and thrombolysis were performed simultaneously to achieve recanalization of the respective pulmonary artery. In all these cases, either Cordis 6F diagnostic catheter or, in the late stages, exchanged with 6F guide catheters were used. The advantages of these procedures are a reduction in the contrast volume load and easy availability of catheters for the same. The 5 mm coronary balloons are commonly available, and if higher balloons are required, exchange with larger balloons was used during the procedures. When a balloon size >5 mm is required, the 6F guide catheter is used. Other advantages of this method include the early withdrawal of the catheter after the procedure. When a thrombolysis catheter is placed for a longer duration in the pulmonary artery, arrhythmias, infections, and thrombosis tend to occur, and thereby, the mortality would increase.\n\n5 mm balloons are average-sized, and the pulmonary arteries are in the range of 8 to 10 mm. The advantage of a 5mm balloon is inside the clot, the balloon can be maneuvered safely inside the smaller pulmonary artery branches without difficulty. In the late stages of the interventional procedure, when the clarity of the distal pulmonary artery or the target artery is good, large balloons like 6 mm or 7 mm Sterling balloons can be used for this purpose. Larger balloons may result in dissection, spasms and proximal dislodgement of clots which may be detrimental. Hence, being ‘average’ in approach may yield better results in uncertainty as definite treatment approaches are not well defined for this clinical condition. The raw data is available in the data availability section below. The number of fluoroscopy and cine acquisitions can be significantly reduced in future similar interventions, and this will reduce contrast usage indirectly.\n\nMany treatment methods are available for physicians to choose for patients with acute pulmonary embolism.14–17 Commonly used methods are systemic thrombolysis, low dose thrombolysis, catheter-directed thrombolysis, ablation of thrombus, Inari flow retriever method, ultrasonic treatment and other embolectomy devices available for this purpose. Though consensus statements are available about the treatment strategy, the choice varies based on the scenario. The scenario of the patient, the clinical manifestations, severity, and co-morbidities like recent surgeries determine the clinicians’ decision.18,19 Also, a classification of the sub-massive/massive differentiation at the bedside is not always possible easily, even though various criteria exist for such a classification.14,18,19 Catheter-directed interventions are especially suited for individuals where the systemic thrombolysis is contraindicated or at high risk for bleeding manifestations. Surgical treatment options have been explored with good results. However, surgery is associated with long bypass time, and the need for a ventilator precludes this method as a routine and easy treatment option.20,21 Contrast reduction will give advantages in these patients as renal failure is commonly associated in an overt or subtle form in these patients. Reducing contrast load and fluoroscopy times are very useful in coronary and peripheral angioplasties.22,23 Renal failure, coronary artery diseases, cancer, and diabetes are commonly associated in patients with deep vein thrombosis.19 Simultaneous balloon dilatation and thrombolysis would be better for greater penetration of the thrombolytic agents and concurrent mechanical clearance of the clots, and the hardware is readily available in all cardiac catheterization labs.\n\nUsing an IVC filter in patients with deep vein thrombosis or venous thromboembolism poses many dilemmas. Metanalysis and NICE guidelines indicate the benefits of the IVC filter in these patients. In the current case discussion, in the opinion of the author, an early deployment of an IVC filter would have benefitted patient-4 by preventing the recurrence of pulmonary thromboembolism.24 It was the practice of the author to insert an IVC filter after a few days or before the discharge of the patients, as some patients would require reintervention of the pulmonary artery. In this case series, all four patients were males. The incidence of pulmonary thromboembolism is higher in the female gender,25 and the treatment availability and outcomes tend to be worse among females.25–27 Hence, this method needs to be evaluated in a large patient population, including the female gender, for a better assessment of the results. These observations were made in a few numbers of patients only. Further studies, including a large number of patients with acute sub-massive pulmonary embolism, needs to be performed for validation of the current observations.\n\n\nConclusion\n\nBalloon angioplasty/maceration and thrombolysis simultaneously can be used as a treatment option in patients with acute sub-massive pulmonary embolism using coronary balloons and catheters. If larger balloon dilatation is required, 6F guide catheters can be used.\n\n\nAuthor contributions\n\nMCA performed the procedures, and wrote the paper.", "appendix": "Data availability\n\nMark Christopher Arokiaraj. (2023). Treatment of Acute Sub-massive Pulmonary Embolism with Balloon Angioplasty and Thrombolysis Simultaneously (Version 1). Zenodo. DOI: https://doi.org/10.5281/zenodo.8159042.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\n\nReferences\n\nAndersson T, Söderberg S: Incidence of acute pulmonary embolism, related comorbidities and survival; analysis of a Swedish national cohort. BMC Cardiovasc. Disord. 2017; 17(1): 155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuppert A, Lees M, Steinle T: Clinical burden of venous thromboembolism. Curr. Med. Res. Opin. 2010 Oct; 26(10): 2465–2473. PubMed Abstract \| Publisher Full Text\n\nScheres LJJ, Lijfering WM, Cannegieter SC: Current and future burden of venous thrombosis: Not simply predictable. Res. Pract. Thromb. Haemost. 2018 Apr 17; 2(2): 199–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCallese TE, Moriarty JM, Maehara C, et al.: Cost drivers in endovascular pulmonary embolism interventions. Clin. Radiol. 2023; 78(2): e143–e149. PubMed Abstract \| Publisher Full Text\n\nWatchmaker JM, Goldman DT, Lee JY, et al.: Increased incidence of acute pulmonary embolism in emergency department patients during the COVID‐19 pandemic. Acad. Emerg. Med. 2020; 27(12): 1340–1343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalra R, Bajaj N, Lancaster WJ, et al.: Efficacy and safety outcomes of catheter-based intervention for treatment of acute severe pulmonary embolism: A comprehensive meta-analysis. J. Am. Coll. Cardiol. 2015; 65(10). Publisher Full Text\n\nJimenez D, Martin Saborido C, Muriel A, et al.: Efficacy and safety outcomes of recanalization procedures in patients with acute symptomatic pulmonary embolism: systematic review and network metaanalysis.2018; 73: 464–471. Publisher Full Text\n\nBrit L, Micheal AD: Are recanalization interventions effective and safe in the treatment of acute pulmonary embolism? Ann. Emerg. Med. 2019; 74(5): 713–716. Publisher Full Text\n\nEinarsson F, Sandström C, Svennerholm K, et al.: Outcomes of catheter‐directed interventions in high‐risk pulmonary embolism‐a retrospective analysis. Acta Anaesthesiol. Scand. 2020; 65(4): 499–506. Publisher Full Text\n\nKhanna NN: Catheter directed thrombolytic therapy and endovacular interventions for acute massive pulmonary embolism. J. Indian Coll. Cardiol. 2016; 6(s1): 153–158. Publisher Full Text\n\nLiang NL, Chaer RA, Marone LK, et al.: Midterm outcomes of catheter-directed interventions for the treatment of acute pulmonary embolism. Vascular. 2017 Apr; 25(2): 130–136. Epub 2016 Jul 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunakata R, Kurihara O, Okazaki H, et al.: Prompt admission and intervention significantly reduce in-hospital mortality for acute pulmonary embolism. Can. J. Cardiol. 2015; 31(10): S93. Publisher Full Text\n\nBloomer TL, El-Hayek GE, McDaniel MC, et al.: Safety of catheter-directed thrombolysis for massive and submassive pulmonary embolism: Results of a multicenter registry and meta-analysis. Catheter. Cardiovasc. Interv. 2017; 89(4): 754–760. PubMed Abstract \| Publisher Full Text\n\nPruszczyk P, Klok FK, Kucher N, et al.: Percutaneous treatment options for acute pulmonary embolism: A clinical consensus statement by the ESC Working Group on pulmonary circulation and right ventricular function and the European Association of Percutaneous Cardiovascular Interventions. EuroIntervention. 2022; 18(8): e623–e638. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTice C, Seigerman M, Fiorilli P, et al.: Management of Acute Pulmonary Embolism. Curr. Cardiovasc. Risk Rep. 2020; 14(12): 24. Epub 2020 Oct 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeller K, Hobohm L, Ebner M, et al.: Trends in thrombolytic treatment and outcomes of acute pulmonary embolism in Germany. Eur. Heart J. 2020 Jan 21; 41(4): 522–529. PubMed Abstract \| Publisher Full Text\n\nPaul JD, Cifu AS: Management of Acute Pulmonary Embolism. JAMA. 2020; 324(6): 597. Publisher Full Text\n\nDudzinski DM, Giri J, Rosenfield K: Interventional treatment of pulmonary embolism. Circ. Cardiovasc. Interv. 2017; 10(2). Publisher Full Text\n\nGupta A, Day JR, Streiff MB, et al.: Mortality and associated comorbidities among patients hospitalized for deep vein thrombosis and pulmonary embolism in the United States: Results from a Nationally Representative Database. Blood. 2020; 136(Supplement 1): 39–40. Publisher Full Text\n\nQiMin W, LiangWan C, DaoZhong C, et al.: Clinical outcomes of acute pulmonary embolectomy as the first-line treatment for massive and submassive pulmonary embolism: a single-centre study in China. J. Cardiothorac. Surg. 2020 Oct 21; 15(1): 321. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToma C, Khandhar S, Zalewski AM, et al.: Percutaneous thrombectomy in patients with massive and very high-risk sub-massive Acute Pulmonary embolism. Catheter. Cardiovasc. Interv. 2020; 96(7): 1465–1470. PubMed Abstract \| Publisher Full Text\n\nArokiaraj MC: Coronary angioplasty and stenting in acute coronary syndromes using very low contrast volume and radiation dosage improves renal and cardiovascular outcomes. J. Cardiovasc. Emerg. 2022; 8(1): 1–13. Publisher Full Text\n\nArokiaraj MC: Off-label uses of Cordis 6F right coronary diagnostic catheters during peripheral interventions.2023. Publisher Full Text\n\nhttp\n\nJarman AF, Mumma BE, Singh KS, et al.: Crucial considerations: Sex differences in the epidemiology, diagnosis, treatment, and outcomes of acute pulmonary embolism in non-pregnant adult patients. J. Am. Coll. Emerg. Physicians Open. 2021 Jan 27; 2(1): e12378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgarwal MA, Dhaliwal JS, Yang EH, et al.: Sex differences in outcomes of percutaneous pulmonary artery thrombectomy in patients with pulmonary embolism. Chest. 2023; 163(1): 216–225. PubMed Abstract \| Publisher Full Text\n\nPribish AM, Beyer SE, Krawisz AK, et al.: Sex differences in presentation, management, and outcomes among patients hospitalized with acute pulmonary embolism. Vasc. Med. 2020; 25(6): 541–548. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "212355", "date": "14 May 2024", "name": "Raja Sekhar Varma", "expertise": [ "Reviewer Expertise Complex cardiovascular interventions" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAcute submassive pulmonary embolism (1) is a known clinical high-risk entity where treatment options include systemic thrombolysis (2), catheter directed thrombolysis, balloon maceration of thrombus, thrombus aspiration, thrombectomy via specialized devices, etc (3). Following this, anticoagulation is administered with different agents and different protocols. Catheter interventions with thrombolysis include catheter-directed thrombolysis, Ultrasound assisted catheter-directed thrombolysis, rheolytic thrombectomy with catheter-directed thrombolysis, and combined techniques. Catheter interventions without thrombolysis include aspiration thrombectomy, mechanical thrombectomy, rheolytic thrombectomy, thrombus fragmentation and combined techniques (4). Current guidelines (4) prefer systemic thrombolysis as the treatment of choice in high risk pulmonary embolism, while reserving catheter directed treatments and surgical embolectomy for failed systemic lysis or in patients with contra-indications for systemic lysis. In this article, the author describes a regimen of simultaneous thrombolysis and balloon angioplasty to treat acute submassive pulmonary embolism in 4 patients with good immediate results in all, but with one patient deteriorating later with presumed recurrence. Balloon maceration of massive/sub-massive pulmonary artery thrombi has been described as a life-saving procedure. Catheter directed thrombolysis has been reported with the least mortality in some studies (5). Combining the two options as a 'pharmaco-mechanical' approach in these sick patients offer many theoretical therapeutic advantages. The balloon maceration of the thrombus rapidly re-establishes adequate pulmonary blood flow, enables the thrombolytic agent to penetrate deeper into the thrombus, while the thrombolysis helps in further breakdown of clots and presumably helps in the lysis of the embolized bits too. Also, the local administration of a lower dose of the lytic agent helps to reduce bleeding events as compared to systemic lytic administration. Thus, a synergistic effect of the two treatment options can be envisaged. Though the author has not mentioned the approach to intervention, it may be presumed to involve an internal jugular or subclavian vein approach if the inferior vena cava is involved; or the contra-lateral femoral vein in the presence of unilateral involvement of the ilio-femoral veins. The choice of the lytic agent is of prime importance. It is likely that the fibrin specific newer agents which can be given as a single bolus may be more efficacious in this setting. In the study, 2 patients received Tenecteplase while the other 2 received Streptokinase. There is some concern about the lesser efficacy of Streptokinase as a \"nonspecific\" older generation lytic agent, especially in a country like India where there may be resistance to the agent due to frequent prior streptococcal infections (6). Allergic reactions too occur frequently with streptokinase due to its antigenic nature. Streptokinase needs to be administered as an infusion due to its short half-life, thereby mandating a longer catheter presence in the pulmonary artery. Lower cost and easier availability are potential advantages of streptokinase in developing economies. Lower doses of lytic agents are typically used in the catheter-directed approach. The author has not mentioned the dosages used for streptokinase and it is not clear how the Tenecteplase dose was calculated. The sequence of use of the two options is also open to discussion - balloon maceration followed by local lytic administration is commonly preferred. Current data on the pharmaco-mechanical approach is limited to registries and small case series. Considering the limited numbers, there are no randomized controlled trials comparing this strategy with systemic thrombolysis. Another option that can be combined with this approach is that of thrombus aspiration/thrombectomy. Though there are many options and combinations possible, the limitations in widespread availability and costs of specific devices prove to be stumbling blocks in the universal applicability of combination procedures. Thus, the effective management of acute submassive pulmonary embolism will necessarily have to be in tune with local resources, expertise and experience.\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? 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "11810", "date": "09 Jul 2024", "name": "Mark Christopher Arokiaraj", "role": "Author Response", "response": "All the procedures were performed in the right femoral approach and after initial iliac vein and inferior vena cava visualization to look for clots. The dose of streptokinase was 1.5million units, which is a vial diluted and given as small boluses during the procedure which usually was about 30 to 40 min in both these patients. In both the patients 90% of the above dose was used. There are allergy concerns during streptokinase use. Commonly observed allergies include febrile reactions, shivering and minor occasional fall in blood pressure. In both the patients in which streptokinase was used there was no allergy reactions. Streptokinase was used in two patients, and slowly, the use of streptokinase is becoming obsolete, and it is being replaced by newer generation tissue-specific thrombolytic agents like Tenecteplase. Streptokinase though considered weaker compared to tissue-specific agents, it has the advantages of lesser bleeding manifestations, 1/10th of the cost of newer generation thrombolytic agents, and still works well in myocardial infarctions, though not preferred. There are theoretical concerns about streptokinase antibodies, especially in the Asian population, which can reduce their efficacy. However, the differences are only marginal, and in real-time clinical observations, streptokinase is still used routinely and widely in myocardial infarctions with success in Asian countries. There are allergy concerns during streptokinase use though it is usually minor. Commonly observed allergies include febrile reactions, shivering and minor occasional fall in blood pressure. In both the patients in which streptokinase was used there was no allergy reactions. Changes in the introduction in the article are made based on the comments from the reviewer." } ] }, { "id": "274500", "date": "08 Jun 2024", "name": "Carlos Jerjes-Sanchez", "expertise": [ "Reviewer Expertise Pulmonary embolism and thrombolysis." ], "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 case series of patients with submassive PE undergoing pharmacoinvasive treatment (thrombus angioplasty and pharmacological thrombolysis). The authors should modify the introduction, which, in general terms, is very vague and does not reflect the safety and effectiveness of the approach to these patients. Is there no similar case in the literature? If there is, they should add it. If there is not, they should emphasize it. They should also address that the advanced treatment and phenotype of submassive PE is poorly defined. Authors have to improve case descriptions. They have to include heart rate, respiratory rate, and blood pressure pre- and post-procedure. ECG and Chest X-ray? The author has to describe. Did they have pre and post-procedure biomarkers? Measurements should be characterized (BNP, high sensitivity cardiac troponin, and dimer D). In the first two cases, 35 mg of tenecteplase was used in bolus. What was the rationale for this dose? TNK titulation is according to body weight. Why did they use streptokinase in the other two cases, and what is the rationale for using that dose? The cases do not describe why they decided to use advanced treatment and not only anticoagulation as recommended in the guidelines in submassive PE. Why did they choose to use vena cava filters if the guidelines only recommend their use in case of absolute contraindication for anticoagulation or recurrence despite anticoagulation and the presence of DVT? Additionally, authors have to set a description of VCF procedure T. They should also explain the rationale for the use of clopidogrel in one of the cases. Not indicated in patients with acute PE\n\nIs the background of the cases’ history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? No", "responses": [ { "c_id": "11809", "date": "09 Jul 2024", "name": "Mark Christopher Arokiaraj", "role": "Author Response", "response": "Reviewer's comment: The authors present a case series of patients with sub-massive PE undergoing pharmacoinvasive treatment (thrombus angioplasty and pharmacological thrombolysis). The authors should modify the introduction, which, in general terms, is very vague and does not reflect the safety and effectiveness of the approach to these patients. Is there no similar case in the literature? If there is, they should add it. If there is not, they should emphasize it. Answer: The introduction section has been modified accordingly. The case series comprises four cases wherein the treatment of acute sub-massive pulmonary embolism was performed in a controlled manner effectively, safely and swiftly without any complications during the procedures. Also, coronary balloons and 6F coronary diagnostic catheters were used in the procedures predominantly to simplify the treatment process of this life-threatening vascular event and to prevent complications like sudden cardiac death, cardiogenic shock, and hemoptysis during the treatment procedures. To our knowledge, no similar case reports or series were published in the past. Reviewer's comment: They should also address that the advanced treatment and phenotype of sub-massive PE is poorly defined. Authors have to improve case descriptions. They have to include heart rate, respiratory rate, and blood pressure pre- and post-procedure. ECG and Chest X-ray? The author has to describe. Did they have pre and post-procedure biomarkers? Measurements should be characterized (BNP, high sensitivity cardiac troponin, and dimer D). Answer: The case details and descriptions are added in the respective cases, and more details are included in the discussion section of the manuscript. The details regarding biomarkers and echocardiography have also been added to the manuscript. Newer references are also added in bibliography section. Reviewer's comment: In the first two cases, 35 mg of tenecteplase was used in bolus. What was the rationale for this dose? TNK titulation is according to body weight. Answer: In the first two patients, the Tenecteplase dose was 35 mg, though the weight-adjusted dose was slightly higher – 40 and 45mg. In the past experiences of the author, the two major complications during the treatment of pulmonary embolism by thrombolysis, where the patient's condition goes out of control, are intracranial bleeding and hemoptysis. As the balloon maceration was performed, the fibrinolytic injections were simultaneously injected. When the clot volume was adequately reduced, the injection was stopped (the 'enough' decision), and during the procedures, all the patients had moderate to severe breathlessness, cough, and lower oxygen saturation, which was a setting for hemoptysis. This dosage decision was based on the intuition of the operator. Reviewer's comment: Why did they use streptokinase in the other two cases, and what is the rationale for using that dose? Answer: Streptokinase was used in two patients, and slowly, the use of streptokinase is becoming obsolete, and it is being replaced by newer generation tissue-specific thrombolytic agents like Tenecteplase. Streptokinase, though considered weaker compared to tissue-specific fibrinolytic agents, has the advantages of lesser bleeding manifestations, 1/10th of the cost of newer generation thrombolytic agents, and still works well in myocardial infarctions, though not preferred. There are theoretical concerns about streptokinase antibodies, especially in the Asian population, which can reduce their efficacy. However, the differences are only marginal, and in real-time clinical observations, streptokinase is still used widely in myocardial infarctions with success in Asian countries. There are minor allergy concerns during streptokinase use, though they are usually minor. Commonly observed allergies include febrile reactions, shivering, and a minor occasional fall in blood pressure. In both the patients in which streptokinase was used, there were no allergy reactions. Reviewer's comment: The cases do not describe why they decided to use advanced treatment and not only anticoagulation as recommended in the guidelines in sub-massive PE. Answer: Though the patients were classified as high-risk sub-massive, all the patients had oxygen saturation <90 percent and all the patients had significant RA, RV dilatation, and RV dysfunction to some extent. Hence, in the author's opinion, these patients could potentially deteriorate or develop severe RV dysfunction/pulmonary hypertension subsequently. Also, the risk of sudden cardiac death and hypotension and potential worsening of the clinical condition was impending. Reviewer's comment: Why did they choose to use vena cava filters if the guidelines only recommend their use in case of absolute contraindication for anticoagulation or recurrence despite anticoagulation and the presence of DVT? Additionally, authors have to set a description of VCF procedure T. Answer: Inferior vena cava filters were used in two of the patients, though guidelines do not recommend them for routine use. The studies on which guidelines are based have yet to come to definitive conclusions, and there is also a lacuna in knowledge regarding the use of inferior vena cava filters. The presence of numerous associated comorbidities, complex clinical situations, a lesser number of pulmonary embolism cases compared to acute coronary syndromes, and varied long-term compliance with drugs in the patients are some of the significant differences across the population section, which preclude generalization in treatment. In the author's previous experiences, the inferior vena cava filters are very safe, and we have yet to see a filter occlusion/thrombosis, etc., even when placed above the renal arteries in a few cases of massive deep vein thrombosis. Inferior venacava filter (Cook Celect) was placed in one of the cases. Through right femoral vein approach the filter was taken and placed in infra renal segment of the inferior vena cava after an initial angiogram. Reviewer's comment: They should also explain the rationale for the use of clopidogrel in one of the cases. Not indicated in patients with acute PE Answer: Antiplatelets like clopidogrel or aspirin are not currently recommended in the treatment of acute pulmonary embolism, though some studies have demonstrated benefits in acute deep vein thrombosis. Occasional use of clopidogrel in small doses in this series is prompted by the observations in the treatment of clots in conditions like acute myocardial infarctions. In this series, in two cases, small tirofiban boluses were given of 10ml in each patient as desperate measures, which is learned by the excellent resolution of clot volumes during primary angioplasties with tirofiban boluses." } ] } ]	1	https://f1000research.com/articles/12-1272
https://f1000research.com/articles/13-338/v1	23 Apr 24	{ "type": "Research Article", "title": "Exploring virtual funding committee practices in the allocation of National Institute for Health and Care Research funding: A netnographic study", "authors": [ "Amanda Jane Blatch-Jones", "Cherish Boxall", "Emmanuel Asante", "Katie Meadmore", "Cherish Boxall", "Emmanuel Asante", "Katie Meadmore" ], "abstract": "Background Funding committees, comprising members with a range of knowledge, skills, and experience, are considered integral to the decision-making process of funding organisations for recommending or allocating research funding. However, there is limited research investigating the decision-making processes, the role of members and their social interactions during funding committee meetings conducted both virtually and face-to-face.\n\nMethods Using a mixed-methods design and following netnography principles, the study observed nine National Institute for Health and Care Research programmes funding committee meetings conducted virtually during October 2020 to December 2021; complemented by interviews with committee chairs and members (18 interviews) and NIHR staff (12 interviews); an online survey (50 responses); and documentary analysis. Personal reflections through immersive journals also formed part of the analysis.\n\nResults Three main themes were identified from the observations, interviews, and online survey:\nefficiency of virtual committee meetings\n(importance of preparation, and the role of formality, process, and structure);\nunderstanding the effect of virtual committee meetings on well-being\n(effects of fatigue and apprehension, and the importance of work life balance);\nunderstanding social interactions and engagement\n(levels of engagement, contribution and inclusivity, awareness of unconscious bias and the value of social networking).\n\nConclusions Examining the decision-making practices of one funding organisation across several research programmes, across multiple committee meetings over one year has generated new insights around funding committee practices that previous studies have not been able to explore or investigate. Overall, it was observed that fair and transparent funding recommendations and outcomes can be achieved through virtual funding committees. However, whilst virtual funding committees have many benefits and opportunities, such as the potential to increase membership diversity and inclusivity, and be more environmentally sustainable, more evidence is needed to evaluate their effectiveness, with particular focus on issues of fatigue, engagement, and committee cohesion, especially when new committee members join.", "keywords": [ "Funding committees", "observations", "interviews", "survey", "netnography", "funding organisations", "mixed methods" ], "content": "Introduction\n\nFunding organisations rely on decision-making procedures to support them to make funding recommendations that are effective, fair, and transparent.1 An integral part of the process involves members with a range of knowledge, skills, and experience (often referred to as funding committees or panels) who convene to evaluate and recommend the allocation of research funding. Several assessments and processes are carried out to support and enable funding committee decision-making. For example, using external peer reviewers to offer an impartial, independent review that informs the funding committee process for funding allocation.2,3 Despite the valuable role these committees play to ensure quality, fair and transparent allocation of research funding, there is a lack of empirical evidence on the processes and functions of funding committee practices. For example, Guthrie et al.4 found no studies examining the social processes of funding committees, despite their central role in the funding allocation process. This could be related to the sensitivity and accessibility around the funding allocation processes and procedures of funding organisations (e.g., funding committee discussions and confidentiality of research applications).2,3,5–7 Challenges in gaining access to funding committees to undertake research or through direct observations is also reported in the literature, along with a lack of well-conducted research looking at more than one funding organisation or in more than one particular context (e.g., more than one research grant programme).4,5\n\nIn 2020, the unprecedented global COVID-19 pandemic challenged the conduct of face-to-face funding committee meetings. This resulted in rapid changes to how funding organisations continued the assessment of research applications, whilst maintaining quality, transparency and fairness of their research funding practices. Whilst most of the evidence focuses on committee members scoring and how videoconferencing may influence final decision-making scores of funding panels,2,4,8–10 there is limited literature on the use of virtual online platforms as an alternative to face-to-face meetings. For example, Pier et al. examined the degree of scoring variability across different panels and whether there were differences between videoconferencing to in-person peer review of research proposals, as they did not have access to actual National Institutes of Health (NIH) study designs.9 They found minimal variation on the final scores between videoconference and in-person meetings, which also supported Gallo et al.’s earlier findings that most review outcomes are not affected by the review setting.11\n\nAttempts to understand the social interactions and social dynamics taking place during the decision-making practice of funding committees are complex, and cannot be understood by examining peer reviewer or committee scores alone.11 Gallo et al. conducted a survey with a cohort of biomedical scientists to try and address the gap in the evidence by looking at the influence, quality, and effectiveness of their most recent panel meeting experience (e.g., being either teleconferencing or face-to-face panels). Although some panel members felt there was an unequal focus and limited engagement from unassigned panel members reviewing the research applications, which could lead to or limit the discussion on scoring, and possibly introduce bias, panel meeting discussions were viewed favourably (e.g., in terms of quality and effectiveness) and were perceived to facilitate the recommended funding decision.6,11 However, a limitation to Gallo et al. study was that it only included a survey examining written/text responses. There were no observations of the panel meeting to confirm the individual responses from the survey.\n\nTo contextualise and understand the more subtle and implicit social interactions of funding committee practices, the exploration through surveys or interview methods alone may not be sufficient. Nonverbal cues provide additional meaning and observing the interactions (along with written notes) provides a more coherent and in-depth account of the social and interactional processes at work during online community settings such as funding committee meetings.\n\nThere are a range of approaches used to conceptualise and understand the virtual social environment we now live in such as virtual ethnography, online ethnography, digital ethnography, and cyber ethnography.12–14 What distinguishes netnography from these forms of ethnography is how the research conducted follows a set of defined research tools, using a pragmatic approach, to study the cultural context and contents, including social dynamics, of online communicative acts in a virtual setting.13,15,16\n\nThe National Institute for Health and Care Research (NIHR), the UK’s largest funders of health and social care research, uses funding committees to evaluate research applications and reach a consensus on the research to be recommended for funding. During the COVID-19 pandemic the NIHR ran virtual funding committees in place of face-to-face meetings for their research programmes. Following and using the principles of netnography, we explored, reflected, and investigated the new and changing landscape of NHR funding committee practice (virtual meetings).\n\nThe aims of the study were to explore virtual funding committee meetings in terms of the formal processes such as technology, resources and formality, and the informal processes such as the social interactions, social dynamics, perceptions, attitudes, and expectations. This paper describes a netnographic study on virtual funding committee practices to gain insight into using online forms of communications (e.g., cultural changes), the benefits, challenges, and barriers to using online platforms (e.g., future considerations) and understand the social interactions in virtual settings (e.g., members participation).\n\n\nMethods\n\nDue to the delicate nature of funding committee meetings and the confidentiality around the discussions and not attributing feedback to an individual committee member, netnography was particularly suited to answer the research questions. The methodological approach offered insights into the cultural processes in a virtual space that would not otherwise have been possible in a face-to-face setting. Netnography allows you to observe in an unobtrusive and non-invasive way (e.g., no observer presence is required), and although netnography shares similar foundations, perspectives and practices to ethnography, there are distinct differences in term of research focus, research methods, data collection and analysis.13,15,17 Exploring the nature and implications of the interrelationship between online social experience and how individuals alter in response to these new technologies is the foundation for netnography.\n\nNetnography follows several fundamental stages like other qualitative methodological approaches, that are inclusive of\n\n1) research inquiry (developing and initiating the research topic and approaches to formulate the research questions)\n\n2) collecting the data (gathering the data through observations, surveys, interviews, online mechanisms, and through an immersive (self-reflective) journal)\n\n3) analysing and interpreting the data (ongoing process of decoding, translating, and coding parts and segments of the data to seek narrative and thematic analysis)\n\n4) sharing the research (contextualising and presenting findings in an appropriate form to disseminate the outcomes to the audience it was intended for).\n\nFollowing interactionist principles, it was possible to explore the virtual conversations about how, where and when things were said, from the committee members through to the role of the chair in steering and managing the discussions. This was important for understanding how virtual social interaction and social encounters are different from physically embedded, face-to-face encounters. The study is reported using the Standards for Reporting Qualitative Research (SRQR) checklist (https://doi.org/10.17605/OSF.IO/RZ6VT).18\n\nTo address the aims and objectives of virtual funding committees in terms of technology, resources, social interaction, social dynamics, attitudes, perceptions, and expectations we proposed to answer the following research questions:\n\n1. How do virtual funding committee meetings provide an alternative approach for the recommended allocation of research funding?\n\n2. Was there any impact of virtual funding committee meetings on the decision-making recommendations for research funding?\n\n3. What were the key components and considerations of running and taking part in a virtual funding committee meeting and do they affect members’ experience?\n\n4. How has the use of virtual online technology affected the social identity aspects of funding committee meetings?\n\n5. Were there behavioural, attitude and relationship considerations (and constraints) when conducting virtual funding committee meetings?\n\nSources of information\n\nTo allow for divergent and in-depth interpretation of the online virtual funding committees observation, interviews and a survey were conducted following the guiding principles of netnography.13,15 The interviews and survey were conducted after the funding committee meeting had taken place and the observational material had been obtained. The sources of information were complementary in nature and enabled cross validation of the observational data.\n\nObservations: The virtual funding committees were recorded on the online platform used by NIHR staff to enable and assist in the minutes of the meeting outcomes which are made publicly available on the NIHR website. These recordings were used for observational purposes only and formed the basis of the netnographic study. We aimed to recruit between 8 to 12 funding committees, one committee meeting per NIHR research programme and funding committees were purposefully selected based on availability and programme engagement, commitment, and approval from the Programme Director.\n\nInterviews: We aimed to conduct 25-30 interviews with funding committee members to understand their experience and expectations of a virtual funding committee meeting, and 10-15 interviews with NIHR staff to explore the practical challenges and potential benefits of virtual funding committee meetings. Several factors influenced the number of interviews needed to reach saturation, and methodologically, there is no definitive number to determine when ‘enough is enough’.19–21 Research by Guest et al. and Hagaman and Wutich suggested a range of 30 to 60 interviews for ethnographic studies.22,23 We aimed to conduct a total of 35-45 interviews or until saturation was reached (e.g., reoccurring conversations with respondents did not emerge any new themes and sufficient data were retrieved to address the research questions).\n\nA purposive sample was used to select funding committee members and NIHR staff based on the NIHR research programmes and on the role performed at the funding committee meeting (e.g., chair, clinician, methodologist, health economist and public representative) to ensure breadth of perspective.24 Invitations were sent to committee members and NIHR staff who attended the funding committee and all committee members were also invited to show their interest in being interviewed as part of the online survey. The interviews were recorded for audio and text data purposes only.\n\nSurvey: The survey was sent to all funding committee members included in the observational cohort to gain further insight and understanding of funding committee practice. A link to the survey (including online consent) to participate was sent to all committee members within four weeks of the virtual funding committee meeting taking place. Committee members were given three weeks to respond to the survey, with a two-week reminder, followed by a final reminder three days before the closure of the survey. We aimed to receive a range of between 160 to 240 responses based on the average size of 20 committee members per research programme. The survey was open for each research programme to participate during the period from October 2020 to January 2022 (based on when the committee meeting took place).\n\nDocumentary analysis: All materials provided to funding committee members were collected for analysis and provided a rich source of written text data to complement the material obtained from the online video footage, interviews, and survey. These documents included the agenda, chair’s brief, guidance for committee members including duties of members, funding committee roles and any After Action Reviews (AAR).\n\nImmersive journal: The immersive journal was used to capture reflections, reactions, perceptions and meanings throughout data collection and during data analysis.15 This type of journal writing reflects on the process of doing the research, exploring new ideas, contextualising the data, capturing experiences, and providing extensive detail into the fragments of data. Immersive journals often contain the combination of what was seen but also what the individual experiences. Capturing these reflections allowed the research team to keep a record and provide any provisional thoughts for wider team discussion (see quality assurance section).\n\nIdentification and community sampling selection\n\nAll NIHR research programme funding committees conducted during October 2020 to December 2021 were eligible to participate in the study, including, Artificial Intelligence in Health and Care Award (AI award), Efficacy and Mechanism Evaluation (EME), Evidence Synthesis (ES), Global Health Research (GHR), Health and Social Care Delivery Research (HSDR) (formerly known as Health Service and Delivery Research (HS&DR)), Health Technology Assessment (HTA), Programme Grants for Applied Research (PGfAR), Public Health Research (PHR), and Research for Patient Benefit (RfPB). Each funding committee was classified as a single online community, based on its activity, interaction, size, and research focus.\n\nPiloting\n\nThe netnographic study involved several methods and these were designed, developed, and piloted with a small sample to ensure their appropriateness. The sample consisted of NIHR staff from the application and funding teams, NIHR Patient and Public Involvement and Engagement (PPIE), programme chairs from the NIHR research programmes, and members of the research team. Particular attention was paid to the observation framework used to facilitate the pre-recorded online video footage from the funding committee meetings, questions in the online survey and the interview schedule. Modifications to the pilot were documented as part of the learning process (e.g., research focus and data collection).\n\nThe study was approved by the University of Southampton, Faculty of Medicine Ethics Committee (ID 57541, 3rd August 2020).\n\nObservations: All NIHR funding committees conducted during October 2020 to December 2021 were invited to take part. Only NIHR funding committees that had approval and agreement from Programme Directors and Chairs were included. Pre-emptive opt-out was used for the approval of using the video footage for research purposes only. This was explained in a covering letter accompanying the Participant Information Sheet (PIS) and was sent to the funding committee members and staff two weeks prior to the meeting, or at a convenient time agreed with NIHR staff. Attendees of the meeting had five working days to consider the option to opt-out of the observational study. There were no opt-outs.\n\nObservations of the funding committee meetings were first viewed for immersive purposes only to allow for personal reflections and initial impressions. This was followed up by a more semi-structured process, focusing on key elements noted in the observation schedule paying particular attention to the processes and practice of using virtual online technology as they emerged (and importantly related to the research questions). A passive-observer position was taken (the research team was not present) as this was the most unobtrusive research approach. The online funding committee meeting recordings were deleted once they were analysed.\n\nInterviews: Two interview guides were used for the two groups of participants: funding committee members and NIHR staff. Where possible, interviews with NIHR staff took take place within a week of the committee meeting, and interviews with the funding committee members were conducted in parallel with the online survey. The participants were purposively selected and invited from pre-defined lists (using Microsoft Excel random number generator), sorted by the relevant NIHR programme. Identified committee members and NIHR staff were sent an invitation letter along with the PIS. They were given two weeks to respond, and a reminder email was sent out after one week. If they expressed an interest in participating, they were contacted to discuss the study requirements and a date was arranged to conduct the interview. Where there was a non-response from the invitation, another set of participants were randomly chosen until we had a sufficient number of committee members to interview.\n\nAny committee member who completed the online survey and expressed an interest to be interviewed, was contacted, and included in the study. This enabled greater flexibility and inclusivity for those who may have had additional experiences to share with the research team. The interviews took between 20-60 minutes, with an opportunity for the participant to follow up on any additional points not covered in the survey or interview schedule. Semi-structured, open-ended questions with prompts were used to inform the interviews. NIHR staff and funding committee member interviews followed the same structure although the focus and topics of interest varied.\n\nThe interviews were conducted using Microsoft Teams, or if this was not feasible due to international location or internet connection, Google Hangout and WhatsApp platforms were used. Research data was recorded in the form of audio and visual files where applicable. Verbal consent was gained from all participants prior to conducting the online interviews. Although some participants completed a written consent form, the interviewer read the consent form to each participant prior to the interview to confirm their agreement and approval. This also gave the participants time to ask questions about the interview (ethical approval included both written and verbal consent due to the conducting the interviews online and recording the interviews in this way. Ethics approval from the University of Southampton, Faculty of Medicine Ethics Committee, ID 57541, 3rd August 2020). This study conducted according to the principles expressed in the Declaration of Helsinki). None of the interviews were transcribed and the interview recordings were deleted once they were listened to, and notes were taken (and as part of the immersive journal).\n\nSurvey: The survey for funding committee members was sent within four weeks of the virtual funding committee meeting and included closed and open-ended questions and Likert scale responses (a total of 16 questions, with 5 follow up questions). The participants were given three weeks to respond to the survey with two follow up reminders (a two-week reminder, followed by a further reminder three days before the closure of the survey). We anticipated the online survey would take approximately 15-20 minutes. Online consent was required from all who completed the survey. The online survey was hosted on a University of Southampton server and participants could access the survey from anywhere that had an internet connection.\n\nAs the study included several methods and approaches, these were drawn on to analyse and interpret the concepts and constructs of virtual funding committees. Both qualitative and quantitative data arising from the study were complementary in nature (rather than competitively) and integrated analytical and interpretative data operations simultaneously.13,15,25\n\nAll qualitative materials (including text data from the virtual observations) were analysed using an inductive approach, allowing the data to drive the thematic coding. Microsoft Excel (Microsoft 365 Version 2308 16.0) and Nvivo software (version 1.6.1) were used to analyse the data, where appropriate. Online survey results were downloaded and initially analysed using a Microsoft Excel spreadsheet, which were later imported into Nvivo to enable cross validation with the observational and interview data (A free alternative Computer-Assisted Qualitative Data Analysis Software (CAQDAS) to using Nvivo is Microsoft Excel or QualCoder that is open source (https://qualcoder.wordpress.com/)).\n\nThe authors independently analysed the data collected from the observations, interviews, and open-ended questions from the survey, which informed the development of the initial themes. The themes were categorised, analysed, and compared across the three data collection approaches. The initial coding and categorisation of the data provided key headlines to help establish and develop the main themes. Within each of the main themes, sub-themes were used to represent the range of topics that were extracted across the three main data sources (observations, interviews, and online survey). The themes and categorisations were independently extracted for each of the data collection methods, and then reviewed by the research team to determine where there were commonalities between what was observed, what was spoken through interviews and what was reported in the online survey. The teams’ immersive journaling also formed part of the verification steps and consensus on emerging themes. Translating the data and seeking consensus and agreement from the research team took place simultaneously during data collection and amendments to categorisations or themes were recorded in Nvivo for transparency purposes.\n\nQuality assurance\n\nIn all qualitative research there is a question around the potential for researcher bias. Due to the research team’s experience and background in the allocation of research funding from the NIHR, there was the potential for preconceptions and biases to occur. To minimise researcher bias, there was more than one researcher on each type of data collection (e.g., interviews, online survey, and observations) to either double code or to review and discuss the preliminary analysis. The research team was also encouraged to keep an immersive journal noting down any reactions, feelings, and readings from the observations, which were used to discuss different perspectives and understand any potential unconscious biases. The research team was therefore confident that this helped to minimise individual and group bias. This was led by the lead researcher to ensure continuity across the study.\n\nFor data processing, several approaches were used to not only process the data but also to maintain quality assurance measures of the collected data in the study. The large volume of data consisted of audio, videos, transcripts, text data, immersive journal notes and survey data, which were imported and held in Nvivo software (https://lumivero.com/products/nvivo/) and Microsoft Excel (accessibility to also analyse and code all data in one location by transferring the data from Nvivo). Combining the data allowed for more divergent thinking and allowed for meaningful interpretation from different sources of data collection, including the verification and validation of the research claims from the observational data. Collecting data using different approaches also allowed for greater interpretation that would not otherwise be possible from the observational data alone.\n\n\nResults\n\nA total of six of ten NIHR funding programmes agreed to take part in the study and nine funding committee meetings during the period of October 2020 to December 2021 were included in the study. Having two NIHR programmes include more than one meeting provided the opportunity to explore variations over time through the observations but also through the follow-up interviews. The online survey was active from October 2020 and closed four weeks after the final committee meeting (January 2022). This allowed all committee members from all committees to participate in the survey during the four-week timeframe after the committee meeting took place. Fifty responses were received from a total of 222 invited respondents (response rate of 22.5%). An invitation to participate in an interview was sent to 60 committee members. Eighteen interviews were conducted with funding committee members across the nine funding committees and included a range of committee roles such as public contributors, statisticians, health economists, clinicians, funding committee Chairs and NIHR programme directors (response rate of 26.6% (16/60) from the invitation to participate, and two from self-selection from the online survey). Twelve interviews were conducted with NIHR staff who participated in one of the nine funding committees and included senior research managers, research managers and assistant research managers (response rate of 44.4%, 27 invitations were sent to NIHR staff).\n\nThe demographic characteristics of the participants involved in the study and the online survey were not collected due to confidentiality. There was wide coverage across all groups of committee members ranging from patient and public representatives to health economists. To further prevent the possibility of individual exposure a high cloaking level was taken across all forms of data analysis and verbatim quotes were amended to remove any associations with funding committee members. All quotes (written and verbal), used from the three sources of data collection, were therefore labelled as P1 (survey), P2 (interview) and P3 (observations).\n\nThree main themes, each with two or three subthemes, were extracted across the three main data sources (observations, interviews, and online survey). Data also revealed a range of experiences between the NIHR funding programmes, but this was not explored further due to potential exposure of participating committee members. Figure 1 illustrates the three themes followed by the sub-themes. It is important to note that it was common for participants to make comparisons with face-to-face funding committee meetings during the interviews and survey, and whilst these comparisons are reported they were not observed directly.\n\n1. Efficiency of virtual committee meetings\n\nThe three themes from the study are highlighted showing the subthemes underneath, representing the data from the observations, interviews and online survey.\n\nThe function and structure of virtual funding committee meetings were a key consideration for all respondents, particularly around the duration of the meeting and the effort required to prepare and run these meetings virtually (particularly for NIHR staff and the Chair). A majority of respondents (37/50, 74%) felt that you could achieve the same outcome through online virtual funding committees compared to face-to-face meetings, and 94% (47/50) of respondents felt that virtual committee meetings have a role to play for the future allocation of research funding (see Table 1).\n\n“Yes, most of the process is the same. You can still read the projects beforehand and score them as it was done before. The discussion is the same and the decision can still be made in the same way using technology (e.g., to vote).” (P1)\n\n“I’ve now attended two virtual meetings and they went much better than I expected. I had worried my participation would be negatively affected from being on a screen all day but that doesn’t appear to have happened. I feel that I’ve been able to contribute fully as if it were an in-person meeting.” (P1)\n\nIn addition, although 86% (43/50) of respondents felt that there were potential opportunities and/or benefits to using virtual online platforms to allocate research funding, 72% (36/50) also felt that there were potential barriers and/or challenges (see Table 1). Thus, funding organisations need to carefully consider the potential trade-offs of conducting funding committee meetings virtually.\n\nIncluded in the benefits of virtual meetings, multiple respondents highlighted the cost and environmental benefits to running these committees virtually and this was a strong motivator across respondents for using virtual platforms for committee meetings in the future. For example, “efficient use of time, travel, environmentally more friendly – better for inclusivity and diversity.” (P2) By contrast, fatigue and social disconnectedness was frequently mentioned by both committee members and NIHR staff as the main challenges with running these committee meetings virtually. For example, “Of all of those fears - gave it a thorough test out, it worked really well. There was no residual doubts. It does work, it’s a different challenge … some of the anxieties are unfounded. It’s physically and mentally exhausting … mentally more demanding.” (P2)\n\nThe level of appreciation for the preparatory work in coordinating virtual committee meetings was noticeable by all committee members and NIHR staff. Having reservations about conducting committee meetings virtually were a concern for committee members (including the chairs), which resulted in several preparatory steps by NIHR staff to support attendees and alleviate any known anxieties.\n\n“You’ve got to really really sing the praises of the secretariat here I mean, they gave it a huge amount of thought beforehand and they tested it out and they made sure me and XX and other senior members were prepped […] it was a real testament to them, the secretariat, that they anticipated what the problems were and simplified things to a point that busy people could interact and they didn’t stick to what was convenient to them. They took on board the kind of feedback and simplified it.” (P2)\n\nAs noted by the NIHR staff there was a tremendous amount of work undertaken prior to the meeting, such as testing, piloting, carrying out pre-runs of the committee meetings, working out how to use the virtual platforms (for conflict of interest on applications) and voting systems. The amount and level of prior preparation for virtual committee meetings was frequently mentioned by NIHR staff and all eventualities were considered to try and prevent any delays during the actual committee meeting.\n\n“We do one re-run to make sure everything works together. Use Teams, but the presentation is on a different package, voting is on another. So we need to make sure everything works together to run but also be in sync with one another. Lots of preparation required especially when there are updates to the platforms prior to meetings, so this is intense for the secretariat staff.” (P2)\n\nFor some NIHR programmes, there was an added level of complexity, as some committees had new members (or were a new committee group) and some committees included international members which meant time zone differences needed to be considered during the preparation. To accommodate new membership, the NIHR staff prepared and run induction and introductory sessions prior to the committee meeting, so that members were familiar with each other and how the committee meetings are run. The chairs were also involved in these sessions, as there were also new chairs and deputy chairs running the committee meetings as well as conducting them virtually. These challenges can have an additional impact on time keeping and trying to maintain the order of the agenda.\n\nAlthough, the set up and preparation involved more staff and more time, there was general consensus that it worked well and met the needs and purpose of running committee meetings: to make recommendations to fund research.\n\n“The most important thing is quality and decisions, we have decades of doing it face-to-face, we understand that approach, we have a relatively limited, forced into it limited exposure to virtual. So far so good” (P2)\n\n“We are now seriously talking about now having more meetings as actually through choice making most of those meetings online so I think clearly if it hadn’t worked, we wouldn’t be having those conversations. So, I think that is a testament to how well it’s worked.” (P2)\n\nIt was clear that virtual committee meetings are conducted differently to face-to-face meetings for several reasons, which need to be considered if virtual platforms are considered in the future. For example, one interviewee commented that virtual meetings were “less free flowing and more structured.” However, it is important to note that NIHR staff followed the same structural format in terms of paperwork, reviewing, preparation and decision-making. The process to decision-making didn’t change, only that it was conducted online rather than in a room face-to-face.\n\nProviding clear structure at the beginning of the committee meeting helped to prepare committee members on what was required of them, the order of the discussions and to ensure all attendees contributed to the discussion. The intensity and increased number of staff required to run these meetings virtually was recognised by both committee members and NIHR staff. For all committee meetings, more NIHR staff were required to ensure every aspect of the meetings ran smoothly, and in some instances NIHR staff also had to manage separate online chats to coordinate and manage the running of the committee meeting on Microsoft Teams or Zoom. However, similar to preparation for meetings, the challenges of virtual meetings became less as more experience was gained from conducting these committee meetings virtually. Several respondents highlighted how the process and structure became easier second time round, as they knew what they were doing.\n\n“Now we are in the flow of the meetings, it’s the new normal and that’s the way it is and we kinda know what we are doing and we have had to adapt to it. But we are used to it and it works well, I think a lot of colleagues prefer it this way. There are no other superficial problems to deal with such as the venue with food or a room or the air con. It simplifies the meeting in a lot of ways.” (P2)\n\nIt was noticeable in all committee meetings that there were clear boundaries, guidance and expectations set out at the beginning of the day, to ensure that all members understood how the day was going to be run. Setting out how the meeting was going to be handled was inclusive, with the committees spending time introducing themselves and encouraging members to turn their cameras on when discussing an application or wishing to participate in the discussion. Although this took time out from the committee discussions, it was appreciated by committee members, especially when there were new members or a whole new committee. However, it was also notable that committees who were more established and known to each other, the flow and structure appeared less formal. This did not deter away from the purpose of the committee meetings, rather it provided a more relaxed atmosphere at a very intense and demanding time. There were also notable differences to the formality of the discussions based on the size of the committee, which did affect the running of the meeting virtually in terms of technology and ability to have cameras on for a committee size of seven compared to 29.\n\n“I did wonder about new committee members joining […] so at the moment we got a committee that knew how the face to face ran and know each other, and how that might play into things moving forward making sure that people are supported coming into this format if it continues.” (P2)\n\nAlmost all committees used the Vevox system (https://www.vevox.com/) to produce the final scores on each application (identical system used in face-to-face committee meetings). The use of the software was extensively tested prior to the committee meeting, and all committees tested the software at the beginning of the meeting (e.g., all members could access and cast their vote, and the results could be presented on screen for committee members). Although there were some glitches across all committee meetings, with varying ways to resolve these issues, it did not appear to impact the overall decision-making for funding recommendations. All chairs provided clear guidance around the threshold scoring, which was universal across all committee meetings.\n\nFor most committee members, the voting system was familiar to members, so despite some technical issues, respondents were encouraging about its use. For those that did not use Vevox for scoring, other methods included using an excel spreadsheet and individual committee members scores being vocally given at the end of the application. The size of the committee and how well established they are was factored in during the preparatory work for the committee meeting and to determine the appropriate approach for scoring. All the scores, for all programmes were collated by an NIHR staff member and all applications when ranked were discussed.\n\nConflict of interests were given adequate time and attention during the preparation of the meeting, particularly for the order of applications. This becomes even more important when running committee meetings virtually but even more so when committees included members from different geographical locations (e.g., international locations). Unlike face-to-face meetings, where committee members leave the room, this is more complex to manage virtually. A similar process to deal with conflict of interests was used by all committees, although there were variations between different virtual platforms due to the functionality options available at the time. Over time, this also meant virtual platform updates enabled more options for NIHR staff and the committee to consider as part of the planning prior to the committee meeting.\n\n“So, whatever I am saying now is based on having run it twice, whereas things may change, the software may change we have already found between the first and second one that some of the functionalities are improving the more we do these remote…we are just running them remotely as general working.” (P2)\n\nFor some committees it was challenging to manage the conflict of interests, which meant that the NIHR staff needed to be fully engaged to ensure all those with conflicts did not return to the meeting until the scoring outcome was removed. Conflict of interests were a particular concern if comments were written in the chat function as everybody, including those who had left the meeting, can see the comments made. Decisions were made early on by the NIHR staff, that the chat functionality was not to be used for comments, only to raise a hand, inform the committee they were stepping away from the meeting, or would like to contribute to the discussion. Over time, there were adaptations to the process and some committees reviewed the process and made relevant changes to accommodate members and make it more streamlined.\n\n“It is trying to make the whole experience easier and better for them in every round that we do.\n\nSome committees are more adept to changes than others, it’s not that they are resistant to it, it’s just the nature of the committee and they are not able to move as quickly as some of the other committees. We have to be mindful of that as well.” (P2)\n\nAs mentioned by respondents, the chairs’ role is challenging and requires different skills in the virtual environment. Different chairs had different styles of chairing and contributed to different levels in the discussion. However, all chairs frequently reminded committee members about the review and decision-making process of the committee, enabling constructive feedback around what was typically referred to as ‘fixable flaws’, ‘fixable faults’ or ‘fundamental flaws’, “could you go through any fundamental issues and anything that could be fixable please?” (P3). Whilst sometimes the transition between chairs wasn’t always smooth, each chair encouraged all members to contribute to discussion. It was suggested and observed that the quality of the chairing made the discussions what they were.\n\n“Chairing these things effectively - you don’t know how difficult it is…a good chair makes it look easy…chairing is hard work.” (P2)\n\n“Good chairing helps to ensure that people are able to ask questions and make comments, although discussion dynamics are much easier in person, when you can see everyone in the room.” (P1)\n\n\n\n2. Understanding the effects of virtual committee meetings on well-being\n\nAs raised in theme one, adopting and changing a critical part of the decision-making process for the allocation of funding due to the COVID-19 pandemic has allowed for alternative approaches to be used. Most respondents have adopted and embraced the changes however, it is important to understand that not everybody is comfortable with virtual environments, and the effects of virtual meetings on well-being raises important considerations around the future recruitment and retainment of committee members. Committees are made up of a diverse group of individuals, and what may work for some may not work for others, and this can have important repercussions for committee members not feeling fully inclusive or equal to other members of the committee.\n\n“Given that it worked, and I suspect that it will continue to work, I think it’s really a different style of solicitation of views, quite a few people could find it easier, Designated Committee Members find it easier talking into a machine. Could be easier for some but harder for others.” (P2)\n\nIn earlier committee meetings, there was apprehension about how it was going to work, and it required extensive preparation and resourcing to try and have a plan for every eventuality. As a result, the planning and preparation, alongside attempts to resolve any uneasiness from committee members, resulted in fatigue for NIHR staff.\n\n“We had worked out a way for delivering them face-to-face and therefore there was some anxiety when we were forced to do them online, it was either online or not do them and not doing them was just incomprehensible, you just couldn’t work out what would happen if we didn’t do them.” (P2)\n\nThis was also coupled with the fact that the meetings were conducted over two or three long days, and often over ran which meant reducing the scheduled break time or continuing until after the proposed finish time. All participants (NIHR staff and committee members) acknowledged that these meetings were always intense and required significant work, however, it was felt that participation and contribution to virtual committee meetings presented different challenges and issues than those experienced at face-to-face meetings.\n\n“It is tiring working online for two consecutive days and one is less able to concentrate to the same degree and over the same period of time as in the in-person meetings. It is easier to disengage.” (P2)\n\n“As chair I found it physically and mentally exhausting - more than face-to-face – it’s more demanding online - as you are not getting all of the visual cues. It’s not the same thing, some are out of focus, poor signal.” (P2)\n\nFor example, respondents highlighted several key concerns such as eye strain from staring at a screen all day, back pain from sitting looking at a screen in one position for longer, and mental fatigue from additional demands in keeping up with conversations online. These issues often made it challenging to be attentive for the whole duration of the meeting.\n\n“The agenda needed to be more realistic, with more time for breaks. I had serious screen fatigue!” (P1)\n\nDue to this, some participants reported that virtual meetings had more challenges and longer-term implications for their wellbeing. For others, virtual meetings were helpful, and for one participant who was hard of hearing commented:\n\nOne issue that is surprisingly better for me - against expectations – […] If you can’t hear someone on a virtual meeting you just say speak up and people do, automatically! (P1)\n\nAnother compounding factor on tiredness and fatigue was the duration and frequency of breaks. Although NIHR staff built in breaks and tried to allow for more breaks, compared to face-to-face meetings, they did not always happen as often the virtual meetings were over-running and so breaks were sometimes delayed or cut short. In addition, participants commented that breaks in virtual meetings are different from those experienced at face-to-face meetings. For example, at face-to-face meetings the drink and food is prepared for you, whereas in virtual meetings participants had to factor making food or drinks into the break time. It was felt that this resulted in less free time or break from their screens.\n\n“… it’s a long time to concentrate. You have to prepare your own food, so break time is down time to quickly prepare food and run back to your desk. It’s harder in that respect, as longer breaks would be better.” (P2)\n\nSome committee members also felt that they got more breaks in face-to-face meetings as often they would have to step out of the meeting because of conflicts of interest. In the virtual environment, participants reported that they could never step too far away from the computer as they were not sure when they might be called back in again and so were always on alert.\n\nAll respondents appreciated there were benefits and challenges associated with virtual committee meetings. For most it was welcomed due to not having to travel and stay overnight, no early mornings or late evenings, taking additional time away from other work or family commitments, ease of participation and more efficient use of time without jeopardising the process.\n\n“… you can sleep in your own bed, you don’t have a thousand-mile round trip, because that is tiring in its own right.” (P2)\n\n“Not having to get up at 4am to make it to London for a meeting and being tired all day. Not having to be away from family and my usual routines. Being able to exercise and eat properly at home and follow my usual routines. Being more relaxed when it wasn’t “my turn”, but still being able to contribute well, listen effectively, and vote.” (P1)\n\nThus, for some, virtual meetings offered greater flexibility to manage a work-life balance, and many respondents indicated that this better accessibility might encourage and/or facilitate more people to become committee members and allow for more diverse membership. However, given the need to move to virtual meetings because of the pandemic, challenges due to all schools and colleges being closed and other restrictions on activities, resulted in additional complexities and requirements during home working. From observing the committees, all members and NIHR staff were sympathetic to the demands placed on individuals and there was regular commentary from the chairs.\n\n“ … what a challenging time we are living in … well done for balancing childcare” Or “… let the committee know if you have to leave by using the chat as we, the secretariat appreciate that working from home means that some of us will have personal commitments to deal with.” (P3)\n\nThere were also comments about virtual platforms allowing a glimpse into colleagues lives that might not otherwise be shared. Although these comments were said in a positive way and participants enjoyed seeing personal backgrounds and pets, for some it added to the blurring of home and work life which they preferred to keep more distinct.\n\n“… one of the nice things about virtual are seeing other people’s backgrounds - is fascinating; and produced a new angle on getting to know people.” (P2)\n\n\n\n3. Understanding social interactions and engagement\n\nThroughout the observations and responses from the survey and follow up interviews with respondents, it was evident that engagement, inclusivity of members, and contribution to the discussions were prominent areas of consideration for virtual committee meetings.\n\n“Structure and pace of the meeting was excellent, and it was very well chaired. Keeping to time and encouraging contributions from all members of the committee.” (P1)\n\nAlthough for many virtual meetings were not a new concept, for some there were practical challenges associated to having the committee meetings virtually. This was particularly relevant for those that did not have the space or sufficient computer equipment such as having two screen monitors or a good internet connection. These issues were seen as being disruptive and problematic, and meant engagement and conversation was at times challenging.\n\n“Very much dependent on internet - so when there is a delay it can feel awkward, especially for voting. Some people do not like the longet silences and find it hard to cope with.” (P2)\n\nThe committee meetings observed showed that there was good quality discussion with input from a variety of members (and equal opportunities provided to contribute to the discussion), providing diverse discussion and indicating member engagement. However, judging the engagement levels in virtual meetings is difficult, especially if someone has their camera off. Indeed, there were challenges around having cameras on for some committees and although NIHR staff and the chair tried to encourage members to have their cameras on whilst presenting or joining the conservation, there were technological issues associated to this.\n\nThose that were on camera were not always looking at the screen. Although some committee members indicated that this was to reduce eye strain or because they were looking at another screen or writing notes, this sometimes gave the impression that they were not engaged with the discussion. Interviews and survey responses also indicated that committee members felt it was harder to concentrate for long periods of time during virtual meetings and they had more distractions to contend with in the home setting.\n\n“… it’s mentally more demanding…it’s more difficult to keep the conservation going … in a room you can pick up on the visual cues, you can’t do that online.” (P2)\n\n“If in face to face you know that you are committing a whole entire day to be there so out of office is on, don’t check emails etc BUT in virtual you can get distracted by emails more easily; virtual gives you flexibility to switch on and off and so may lose concentration.” (P2)\n\nFurthermore, because of the virtual environment, some members were at work or had just arrived home from a day’s work (this was particularly true for non-European countries and the time zone variations). Although this demonstrated inclusivity and allowed diversity of membership, this added an extra layer of distraction and fatigue to these members which may have had an impact on their level of engagement during the committee meeting. For example, attendance at meetings with international members was often found to drop off as the day went on due to different time-zones.\n\nHaving cameras off was found to make it difficult to read social cues and body language. The value and importance of ‘reading the room’ for some was just as important as the conversation itself, and some felt that this social connectedness was totally missing through virtual platforms.\n\n“You do have some visual cues online but it’s just not the same as being in a room, the visual cues are just different. The quality of the cameras vary, the connections vary.” (P2)\n\n“When you’re in a room there is a way to negotiate time, in a sort of untold way by using visual cues and looks and all kinds of things you can do to create your space. This is missing completely.” (P2)\n\nThis was found to be particularly challenging for chairs because it was harder to know if further discussion was needed or if the committee were generally happy with the decision. Chairs encouraged inclusivity but found it more difficult to ensure they were providing sufficient opportunity for all committee members to contribute to the discussion in a virtual setting due to the lack of social cues. This was particularly noted as the meeting progressed throughout the day. However, all chairs continuously engaged with the committee, openly providing members the opportunity to contribute, and therefore maintaining a quorum.\n\n“Earlier proposals in the day received wider participation but by the end of the meeting, few people could contribute, understandable as it was a long day.” (P2)\n\n“The breaks were brief, and people were tiring and as the meeting went on more cameras went off and people were becoming less engaging.” (P2)\n\n“I am not sure how to improve the inertia towards the end of the meeting - understandably the members were tired, and the chair and secretariat did an amazing job of ensuring that the last proposals were also discussed in depth.” (P2)\n\nChairs found it difficult to always spot when someone had something to say and how to bring different members into the discussion, noting that ‘it is a lot harder than it looks’. Different techniques were used between programmes and the level of support from NIHR staff varied by committee meeting. In line with this, some members commented that they felt it was more difficult to follow the conversation, to interrupt discussion and to add their contributions during the committee meeting.\n\n“Etiquette is that you keep camera off unless speaking so most chairing is done in the dark.” (P2)\n\n“The introverts who are waiting to contribute and Teams does not do them justice. I am see that in a room but not online - it’s really hard … it’s a big committee and the noisy voices are heard more. So, I have to invite members to talk.” (P2)\n\nThere were differences across the programmes on how long the Designated Committee Members (DCMs) spoke for about an application and what was included in their summaries. However, the purpose of stage 1 applications is different to stage 2, in that stage 1 applications are shorter, and the primary purpose of the committee is to assess the quality and value of the research question. For stage 2 applications, the role of the committee is to make a funding decision based on the full application. From the observations, at stage 1, decision making often relied more on committee member comments and there was less discussion. At stage 2, discussions were fairly varied as members with different roles were assigned as DCMs for each application – e.g., clinician, methodologist and patient and public contributor, so each reviewed the proposal with a different perspective. This resulted in some applications having less or more time allocated, as the chair typically but not exclusively would do a final summary of each application prior to the scoring of the application.\n\n“Pace sometimes quite fast but generally sufficient discussion.” (P1)\n\n“… it is faster paced online, and you need to be quick to jump in.” (P2)\n\nNevertheless, all data sources indicated that moving to virtual does not seem to have had an impact on the overall decision making. It was felt that the virtual setting made the discussion more focused and there was less deliberating over every point. It was also felt that contributions were made by different members, which indicated that committee members were engaged with the process.\n\n“… think that it is highly unlikely that virtual impacts on the quality of the decision, as most of the preparation happens beforehand.” (P2)\n\n“From the quality of the contributions going remotely hasn’t had any impact in terms of engagement. Where I think it’s impossible to make a call is when the most interesting conversations happen when you push it out to the floor … it is infinitely more difficult to do that remotely.” (P2)\n\nUnconscious bias is a function of being human and so it is unlikely that any decision-making by a funding committee will be completely free from bias in whatever format committee meetings are conducted (virtual or face to face) but recognising such biases may be present and acting upon them is important.26,27\n\nUnsurprisingly committee meetings with many members, some behaviours were observed in this case that could potentially reflect unconscious bias. All respondents were clear that it was essential that all applications were given a fair hearing, and overall, this is what was observed in the virtual context. However, as with face-to-face committees, when time was short, or the committees were over running, there were instances when the committee might not spend as long discussing applications with a clear trajectory of a very low or high score.\n\n“Very positive comments indeed. External peer review comments are also very positive so don’t want to spend too long on this.” (P3)\n\nIt was observed that a lot of emphasis was placed on external reviewers and DCMs (three or four committee members who were assigned to review a particular application) scores in the assessment process. This is usual practice, and there was no variation between the virtual setting to face-to-face. All committee members had the opportunity to contribute to discussions on an application. However, there were examples of some mismatch in the scores and written comments given (from the external reviewers and DCMs) and some clustering of scores. For example, written comments might highlight multiple and significant flaws in a study and a score of four is given (which is fundable) or lots of strengths of the application are given and then it wasn’t scored highly. The interviews highlighted that NIHR staff, and some committee members were aware of this issue, in that the committee scores were the only ones considered in the overall ranking of the applications. Pre committee scores can, however, change because of committee meeting discussions as other members raise issues on an application. Chairs would encourage committee members to use the range of scores available1–6 and described what the numerical scores meant at the start of the committee meeting. This was repeated throughout the committee meetings to ensure balance across all applications.\n\nIt was also observed that how things were said and by whom can also contribute to potential unconscious bias by framing an application in a positive or negative light. For example, “beautifully written” or “This is the first time I’ve given a 6; it’s very impressive, one of my favourites” presented applications in a positive light, whereas “eye wateringly expensive”, or “this one is a bit of a marmite application” suggested room for improvement (P3). Some respondents expressed that they felt this happened more with experienced or senior members and in committees that had more established members rather than several new members of the committee.\n\n“When certain people have opinions and say something in an authoritative tone it can become the mood of the room” (P2)\n\n“Perhaps one thing is the virtual space allows for the dominant characters to dominate … There are people who dominate more, and it’s transferred to the virtual space. So, it’s finding ways to deal with this - mechanisms to ensure everyone has a voice using the virtual space as the platform for committee meetings.” (P2)\n\nThere were some instances when committees paid particular attention to an applicant’s gender or career stage. For example, in a summary of an application one committee member noted, “led by two female PIs.” It was felt that this comment was meant as a positive one, by which committee members were paying particular attention to improve research equality, diversity, and inclusion. Another example was when a dedicated committee member stated when introducing an application “this is one where there is a junior PI and I think they provide quite good justification for that.” (P3)\n\nAcross all programmes, the observations found that some members of the committee were referred to by their role in the discussions. Although, often this was done in a factual way or because they needed certain expert advice (e.g., health economics), this was most notable for patient and public contributors. For some respondents, it was also felt that the larger the committee the more opportunities for bias could be introduced.\n\nFrom the observations, it was noted that one NIHR committee was actively engaged in increasing awareness of unconscious bias through the use of a video training. In the second observation of this committee, members were observed to recognise unconscious bias on occasion indicating an increased awareness in this topic. There was also the discouragement of using the chat function as it could have been seen to introduce bias.\n\n‘… this is probably my unconscious bias but …’ (P3)\n\nOne of the biggest challenges with conducting committee meetings virtually was the lack of social interaction and networking opportunities. The importance of social networking was frequency mentioned by members of the committee and NIHR staff. The survey data found that 54% (27/50) of respondents considered face to face networking at funding committees as very important to them and 34% (17/50) reporting it was somewhat important (see Figure 2).\n\nQuestion reported from the online survey “How important is face-to-face networking at funding committees to you?” The responses alongside the percentage for each response is reported using different colours to represent each response in the pie chart. There was no response for the ‘less important’ option as shown above.\n\nA range of social aspects were described by respondents as lacking through having to conduct the committee meetings virtually, which were not entirely related to just the meeting itself. The survey reported that 66% (33/50) of respondents did not consider the additional features of using virtual platforms to conduct funding committees (e.g., chat function, raising your hand, and voting methods) provided more opportunities for members to engage in the committee discussions. Several committee members and NIHR staff also reported missing the chance to socialise during break time and the social gathering and networking after the committee meeting during virtual meetings.\n\n“… we did set out what we wanted to achieve, it was three incredibly long days without the nice sort of nice relaxation at the end … you all go out for a nice meal and relax and talk to the committee members in a more relaxed and social environment. That social aspect was missed and although that social aspect is not vital it’s not what we are there for it oils the wheels.” (P2)\n\nGetting to know other committee members was suggested to help with understanding context for comments from certain members and it was evident that more established committees had more conversational dialogues and light-hearted comments.\n\n“Miss out on the conversations at coffee time - discussions more around general things and helps us to understand where each other comes from. But you still learn from this. You’re able to ask questions for example, about a particular point or method. We can go and talk about it, about what do you think about this. So, it’s the whole, not just the yes no we approve an application, there is more to it than that and we miss out on that. It becomes very process driven, a tick box process in some respect.” (P2)\n\nSocial networking was also suggested to be particularly important for new members to ‘get to know’ the other committee members. For some respondents, it is during these social interactions that collaborations and networks are formed, and this was seen as an important part of becoming a member of a committee. It was also seen as a positive benefit from spending what would ordinarily be non-work time with existing committee members, given the level of commitment required during funding committee meetings. Participants reported that this lack of social and networking engagement for future committee members could impact whether individuals choose to join a committee in the future. These opportunities provide new members with a sense of feeling included and key to being integrated as part of that committee and developing oneself at a personal level. Without these social interactions, for some respondents, there was a loss of a sense of belonging as joining a committee the first time was described as ‘daunting’.\n\n“It is daunting when you’re a new member and social engagement and interaction is key to feeling integrated and part of the committee.” (P2)\n\nSeveral committees encouraged social networking as part of the preparation of the meeting, especially for those committees that were newly established, had several new members or involved several international members. This was received well by new members, although this added additional pressure to the preparation time for NIHR staff.\n\n“… So we try and do an induction, give people the chance to observe and if they can’t do that we will do an induction but maybe that might be something that would be good to keep so they get to know our faces and they get to know that they can come to us still although it is remote ….” (P2)\n\n\nDiscussion\n\nTo our knowledge this is the first study to have explored funding committee practices through observations, complemented with follow up interviews and an online survey with committee members, and interviews with staff who facilitated and organised these meetings. The study also applied a methodological approach that specifically focuses on online social interactions, which offered a unique and in-depth understanding about the social processes of virtual funding committees given their central role in the funding process. Examining the decision-making practices of one funding organisation across several research programmes and across multiple committee meetings over a period of one year has therefore generated new insights around funding committee practices that previous studies have not been able to explore or investigate due to gaining access or sensitivities around the funding allocation processes.2–6\n\nThe findings highlighted the complexities of preparing and running funding committee meetings and also how the meetings, when conducted virtually, introduce new challenges and benefits than those conducted in a different setting (e.g., face-to-face).6,11 The study found that several parameters are not transferable from a face-to-face to a virtual setting, such as timings, location, equipment, and physical attributes. All of which can have wider implications for funding committee members and funding organisations preparing and structuring the committee meetings. Virtual meetings require different functional considerations, such as how to manage conflicts of interest when someone cannot simply leave the room or when someone has a poor internet connection. The findings highlight the level of planning and preparation required by staff to mitigate against these issues along with the repercussions of being reliant on technology, meant that more concentration was required from all staff and committee members, and often resulted in an increased level of fatigue. In addition, scheduled breaks and changes to the standard structure of committee meetings resulted in less free time, further exacerbating fatigue in the virtual experience.\n\nAdjusting to using virtual platforms during the pandemic has shown that over time, committee members and staff have become more accepting of new ways of working, as what was seen as ‘daunting’ in the latter part of 2020, was far less of an issue one year on. Whilst it was evident that running committee meetings virtually had its benefits in terms of work-life balance, travel, and environmental sustainability, it was suggested that this sometimes came at a price. For many, there was a ‘trade off’ with not having the opportunity to socially interact or network whilst attending the funding committee meeting, as well as increased fatigue. The themes highlight how important levels of engagement and social interaction are, especially for new members of the committee and during committee discussions. These findings are in line with previous literature, suggesting that an unequal focus or limited engagement from funding committee members can lead to or limit funding decision discussions.6,11,26 Nevertheless, observations of committees and feedback from committee members agreed that despite discussions being more focused, the decision-making process was largely the same in the virtual meeting.\n\nThe role and function of committee meetings, whether they are virtual or face-to-face does not change, and both have benefits and challenges. It is important to note that some of the challenges reported about virtual committees were also relevant to face-to-face meetings. For example, the potential for unconscious bias was not something unique to virtual meetings and is reported in the literature.26,27 What was evident from the observations was how the virtual committees evolved over time and adapted their approach to accommodate committee members but also to make the process easier to manage (see Table 2).\n\nThe organisational structure of funding committee meetings breaks and timings of the applications need careful consideration, with potential flexibility or options to change the agenda order. This is especially relevant to committees who have members from different time zones. It is also important that processes are in place to minimise potential biases and ensure no power imbalances between different committee members or towards certain applications. As demonstrated by one of the NIHR funding committees observed, one possible consideration that may help with this is for all members of the committee to view an unconscious bias video prior to reviewing applications as part of the preparation process of the funding committee meeting. More broadly, there is also an opportunity for funding organisations to consider a more inclusive and diverse funding committee membership that takes account of differences in time zones, disabilities, part-time work, or those with other responsibilities.\n\nThe process and formality of running funding committee meetings is also imperative to ensure inclusive contributions and engagement for all members of the committee. Encouraging members to have their cameras on is one consideration, although there are challenges associated to this, especially where internet connections are unreliable. There is a need to appreciate how new members interact and engage with existing funding committees to encourage participation and contribution to the discussions. Such opportunities could consider face-to-face development days or a mixed approach to how the funding committees are held (e.g., mixture of face-to-face and virtual meetings).\n\nFinally, future requirements for training and additional guidance to support existing and new committee members and the chair are important considerations. Chairing virtual meetings is different and requires a different set of skills. At times it was challenging for the chairs, who found it difficult due to having limited social cues from the committee to aid discussion, multiple technologies/screens to manage and because the duration of the meetings meant that they had to interact virtually for extended periods of time. This often led to increased fatigue, particularly during the first round of committee meetings. Several options could be considered to lessen the virtual fatigue, including guidance to support committee members and chairs, extended breaks and/or shorter meetings. It is also important to consider how new members of the funding committee could be integrated into existing committees, when the format is predominately virtual. Such considerations could be face-to-face development days or virtual social meetings. In addition, allowing more junior members to join as observers or trainees on a funding committee may encourage diversification of funding committees as a form of training. Thus, virtual funding committees not only have additional training considerations but also the offer the opportunity to be a form of training which in turn may facilitate the diversity of committee membership and increase the transparency around funding committee practices.\n\nThe main strength of the study was the inclusion of nine NIHR committee meetings across several research funding programmes. As we included committee meetings that took place over a one-year period, it meant that committee members and staff had experienced more than one virtual committee meeting. This enabled us to see how the views, opinions and expectations of committee members changed over time. Capturing these experiences had important implications for the findings and how experiences can vary across different funding programmes. As the study included interviews and an online survey, we were able to follow up and support our non-participatory observational claims, which can often be seen as a limitation of netnographic studies. It is also important to note that due to the complexity, structure and formality of funding committee meetings, some areas considered important ran through more than one theme. By using a methodological approach that was based around online social interactions, it was possible to gain valuable insights into the recommendations of research funding allocation, without influencing the views, opinions, or expectations of the committees or staff.\n\nEthical considerations and recommendations of netnographic studies are important to ensure information about users’ identification is kept confidential, which is frequently reported in the literature as a weakness of this type of study.15,28 To overcome this, the study sought ethical approval and had a high cloaking level to avoid identification of the survey respondents, interviewees and members of the committee.\n\nA limitation to the study was that it was based on observations and experiences of funding committee meetings held in the early part of the COVID-19 pandemic (2020-2021) and therefore did not include any comparable data with face-to-face committee meetings. Early insights to the findings of the study and based on committees own experience, some changes to the funding committee process may have already been implemented due to developments and initiatives taking place simultaneously with the current study. Another limitation was around potential researcher bias during analysis and interpretation of the observations, interviews, and survey responses. Four researchers involved in the analysis, each bringing their own experiences and knowledge on funding committee practices, could have produced bias on overall expectations and interpretation of findings. However, actively encouraging to keep an immersive journal throughout data collection and analysis, and enabling regular reflective discussions on reactions, feelings, and observations, helped to minimise bias and maintain a level of autonomy.\n\n\nConclusion\n\nAlthough there are several areas for consideration for continued virtual funding committee meetings, such as inducting new members and maintaining inclusivity for all committee members, the study found that conducting funding committee meetings virtually was feasible and funding decisions continued to be fair and transparent.\n\nGiven that there is no current evidence or use of observations to understand the social processes and functions of funding committee meetings, this study has shown its value and critical contribution to building an evidence-informed approach. By applying a netnography methodology to observe, understand and capture the views of virtual funding committees, it was possible to gain insight to these committees attended by the respondents.\n\nAlthough there is acceptance and a place for virtual committee meetings from committee members and staff, it is important to remember that this is not the view of all members. Whilst virtual funding committees have many benefits and opportunities such as the potential to enable work-life balance, inclusivity for members, reduce costs, and be more environmentally sustainable, more evidence is needed to evaluate the longer-term sustainability of virtual committee meetings in the allocation and decision-making of funded research.\n\n\nEthics and consent statement\n\nVerbal consent was gained from all participants prior to conducting the online interviews. Although some participants completed a written consent form, the interviewer read the consent form to each participant prior to the interview to confirm their agreement and approval. This also gave the participants time to ask questions about the interview (ethical approval included both written and verbal consent due to the conducting the interviews online and recording the interviews in this way.\n\nEthics approval from the University of Southampton, Faculty of Medicine Ethics Committee, ID 57541, 3rd August 2020). This study conducted according to the principles expressed in the Declaration of Helsinkiv.", "appendix": "Data availability statement\n\nIn line with our ethical approval, the interview, survey, and observational data cannot be shared publicly to maintain confidentiality of our participants. The interview, survey and observational guides are provided in OSF: Exploring virtual funding committee practices in the allocation of National Institute for Health and Care Research funding: A netnographic study. DOI 10.17605/OSF.IO/RZ6VT. 29\n\nThis project contains the following underlying material:\n\n• Appendix 1: Observation guide\n\n• Appendix 2: Interview guide for committee members\n\n• Appendix 3: Interview guide for NIHR staff\n\n• Appendix 4: Survey questions SRQR checklist\n\nData are available under the terms of the CC0 1.0 Public domain dedication (https://creativecommons.org/publicdomain/zero/1.0/).\n\nAdditional quotes under each theme are available from the Insight team, School of Healthcare Enterprise and Innovation, University of Southampton (contact corresponding author: A.J.Blatch-Jones@southampton.ac.uk) for researchers who meet the criteria for access to the data.\n\nReporting guidelines: OSF repository. SRQR checklist for “Exploring virtual funding committee practices in the allocation of National Institute for Health and Care Research funding: A netnographic study.” DOI 10.17605/OSF.IO/RZ6VT (https://osf.io/rz6vt/) 29 under the terms of the License: CC-BY 4.0 International license.\n\n\nAcknowledgements\n\nWe would like to thank all participants that took part in the study and for those NIHR staff who contributed to the early design of the study. We would also like to thank the NIHR Research on Research team for reviewing early draft versions of the article.\n\n\nReferences\n\nHug SE, Aeschbach M: Criteria for assessing grant applications: A systematic review. Palgrave Commun. 2020; 6(1): 1–15.\n\nObrecht M, Tibelius K, D’Aloisio G: Examining the value added by committee discussion in the review of applications for research awards. Res. Eval. 2007; 16(2): 70–91. Publisher Full Text\n\nOlbrecht M, Bornmann L: Panel peer review of grant applications: what do we know from research in social psychology on judgment and decision-making in groups? Res. Eval. 2010; 19(4): 293–304. Publisher Full Text\n\nGuthrie S, Ghiga I, Wooding S: What do we know about grant peer review in the health sciences? F1000Res. 2017; 6: 6. Publisher Full Text\n\nHuutoniemi K: Communicating and compromising on disciplinary expertise in the peer review of research proposals. Soc. Stud. Sci. 2012; 42(6): 897–921. Publisher Full Text\n\nGallo SA, Schmaling KB, Thompson LA, et al.: Grant reviewer perceptions of the quality, effectiveness, and influence of panel.2020.\n\nCarpenter AS, Sullivan JH, Deshmukh A, et al.: A retrospective analysis of the effect of discussion in teleconference and face-to-face scientific peer-review panels. BMJ Open. 2015; 5(9): e009138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBohannon J: Meeting for peer review at a resort that’s virtually free. American Association for the Advancement of Science; 2011; vol. 331. : 27. PubMed Abstract \| Publisher Full Text\n\nPier EL, Raclaw J, Nathan MJ, et al.: Studying the Study Section: How Group Decision Making in Person and via Videoconferencing Affects the Grant Peer Review Process. WCER Working Paper No. 2015-6. Wisconsin Center for Education Research; 2015.\n\nBarnett AG, Herbert DL, Campbell M, et al.: Streamlined research funding using short proposals and accelerated peer review: an observational study. BMC Health Serv. Res. 2015; 15(1): 1–6.\n\nGallo SA, Carpenter AS, Glisson SR: Teleconference versus face-to-face scientific peer review of grant application: effects on review outcomes. PLoS One. 2013; 8(8): e71693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFenton A, Parry KD: Netnography–an approach to ethnography in the digital age. The SAGE Handbook of Social Media Research Methods. 2022; p. 214. Publisher Full Text\n\nKozinets RV: Immersive netnography: a novel method for service experience research in virtual reality, augmented reality and metaverse contexts. J. Serv. Manag. 2023; 34(1): 100–125. Publisher Full Text\n\nAddeo F, Paoli AD, Esposito M, et al.: Doing social research on online communities: The benefits of netnography. Athens J. Soc. Sci. 2019; 7(1): 9–38. Publisher Full Text\n\nKozinets RV: Netnography: The Essential Guide to Qualitative Social Media Research. SAGE Publications Limited; 2019.\n\nKozinets RV: Netnography. Handbook of qualitative research methods in marketing. 2006; Vol. 129: 142.\n\nKozinets RV, Gambetti R: Netnography unlimited: Understanding technoculture using qualitative social media research. Routledge; 2020.\n\nO’Brien BC, Harris IB, Beckman TJ, et al.: Standards for Reporting Qualitative Research: A Synthesis of Recommendations. Acad. Med. 2014; 89(9): 1245–1251. Publisher Full Text\n\nBaker SE, Edwards R: How many qualitative interviews is enough.2012.\n\nGuest G, Bunce A, Johnson L: How many interviews are enough? An experiment with data saturation and variability. Field Methods. 2006; 18(1): 59–82. Publisher Full Text\n\nHagaman AK, Wutich A: How Many Interviews Are Enough to Identify Metathemes in Multisited and Cross-cultural Research? Another Perspective on Guest, Bunce, and Johnson’s (2006) Landmark Study. Field Methods. 2017; 29(1): 23–41. Publisher Full Text\n\nBernard HR: Research methods in anthropology: Qualitative and quantitative approaches. Rowman & Littlefield; 2017.\n\nMorse J: Handbook for qualitative research. Lincoln NDY, editor. Thousand Oaks, CA: Sage; 1994.\n\nMiles MB, Huberman AM: Qualitative data analysis: An expanded sourcebook. Sage; 1994.\n\nCostello L, McDermott M-L, Wallace R: Netnography: Range of practices, misperceptions, and missed opportunities. Int. J. Qual. Methods. 2017; 16(1): 160940691770064. Publisher Full Text\n\nGuthrie S, Rincon DR, McInroy G, et al.: Measuring bias, burden and conservatism in research funding processes. F1000Res. 2019; 8(851): 851. Publisher Full Text\n\nSato S, Gygax PM, Randall J, et al.: The leaky pipeline in research grant peer review and funding decisions: challenges and future directions. High. Educ. 2021; 82(1): 145–162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanger R, Beckman SC: Sensitive research topics: netnography revisited. Qual. Mark. Res. Int. J. 2005; 8(2): 189–203. Publisher Full Text\n\nBlatch-Jones A, Meadmore K, Boxall C, et al.; Exploring virtual funding committee practices in the allocation of National Institute for Health and Care Research funding: A netnographic study. OSF. 2024. Publisher Full Text Reference Source" }	[ { "id": "284406", "date": "03 Jun 2024", "name": "Adrian Barnett", "expertise": [ "Reviewer Expertise research funding", "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\nThis is an interesting and worthwhile study to examine panel dynamics using a real process. There was a good balance of data collection from reviewers and staff. It appears to be well planned and well conducted. The findings are likely useful for other funders.\nPerhaps it is out of scope, but there is some debate on whether panels can be removed in favour of independent peer reviewers. As well as the time and money costs of panels, there are concerns that they reduce the variance in scores towards strong voices [1], and that committee members inevitably score proposals which are outside their expertise. Some studies have found that panels are not be useful for decision making [2,3].\nOne anecdotally cited benefit of panels is that they provide extra scrutiny, as reviewers cannot be disengaged, e.g. https://x.com/GillBedi/status/1697876646936928333. Were there any comments on this?\n\"noisy voices are heard more\" – this is the same in a face-to-face meeting: \"some of the more dominant personalities to take over the group and have an undue influence on the grant outcomes\" [1].\n\"the larger the committee the more opportunities for bias could be introduced\", but too small can be seen to not have enough voices [4]. There is an interesting balance to strike here.\nHaving \"networking opportunities\" is in my opinion a negative aspect of panels. It provides an advantage to those who are invited and can attend the meeting. It potentially contributes the Matthew effect in research funding [5], as those who attend the meeting get privileged insights into how to write their next application.\nMinor comments - PIS = participant information sheet? - \"This study conducted according to the principles\" missing 'was' - Pie charts are not a recommended data visualisation, see for example https://scc.ms.unimelb.edu.au/resources/data-visualisation-and-exploration/no_pie-charts - A consent rate of 6 out of 10 committees is disappointing given the aim to improve the process. I realise this was beyond the researchers' control. - The questions shown in Table 1 have a positive slant towards the online format. More neutral wording would have been preferable. -“Earlier proposals in the day received wider participation but by the end of the meeting, few people could contribute, understandable as it was a long day.” – this is likely the same in face-to-face meetings. - A description of the scoring process may be useful background. - \"view an unconscious bias video prior to reviewing applications\" the NHMRC encourage reviewers to do the unconscious bias training (“Harvard Implicit Bias” https://www.nhmrc.gov.au/funding/peer-review). - \"some clustering of scores\" clustering within the scores from the same committee member (e.g., scores for feasibility, novelty) or across the committee? - Typo: \"Helsinkiv\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11948", "date": "03 Jul 2024", "name": "Amanda Blatch-Jones", "role": "Author Response", "response": "Perhaps it is out of scope, but there is some debate on whether panels can be removed in favour of independent peer reviewers. As well as the time and money costs of panels, there are concerns that they reduce the variance in scores towards strong voices [1], and that committee members inevitably score proposals which are outside their expertise. Some studies have found that panels are not be useful for decision making [2,3]. Thank you for the additional references you have cited. One anecdotally cited benefit of panels is that they provide extra scrutiny, as reviewers cannot be disengaged, e.g. https://x.com/GillBedi/status/1697876646936928333. Were there any comments on this? Our findings indicated that the committee engaged in good quality discussion with input from multiple members indicating that members were engaged with the review process. In addition, data not reported in this paper demonstrate that committee members felt that the reviewing, critically appraising and discussing applications was the most cited role of committee members. However, as noted in the paper, engagement throughout the whole meeting was sometimes difficult to gauge as cameras were not always on and members felt there was the potential to be more distracted on virtual calls. \"noisy voices are heard more\" – this is the same in a face-to-face meeting: \"some of the more dominant personalities to take over the group and have an undue influence on the grant outcomes\" [1]. We have added this as an additional example of similarities in face to face and virtual meetings in the discussion (pg. 24) \"the larger the committee the more opportunities for bias could be introduced\", but too small can be seen to not have enough voices [4]. There is an interesting balance to strike here. Thank you for your thoughtful comment, and it is an important point to raise. Our paper reported how we found smaller committees to be more informal than larger committees. We agree that there is a balance to strike, and although there has been some research into ideal numbers of peer reviewers and committee members there is no consensus. Future work could explore this in more detail Having \"networking opportunities\" is in my opinion a negative aspect of panels. It provides an advantage to those who are invited and can attend the meeting. It potentially contributes the Matthew effect in research funding [5], as those who attend the meeting get privileged insights into how to write their next application. We have adjusted the point raised in the discussion to highlight the potential advantage of being a committee member, although also highlighting that virtual meetings could be a way to facilitate the diversity of the committee. Minor comments - PIS = participant information sheet? - \"This study conducted according to the principles\" missing 'was' - Pie charts are not a recommended data visualisation, see for example https://scc.ms.unimelb.edu.au/resources/data-visualisation-and-exploration/no_pie-charts We have changed the pie chart to a more suitable diagram. - A consent rate of 6 out of 10 committees is disappointing given the aim to improve the process. I realise this was beyond the researchers' control. As you point out the consent rate was unfortunately out of our control. However, having included two programmes with more than one committee provided us with the opportunity to explore changes over time, which is a unique feature to the study. - The questions shown in Table 1 have a positive slant towards the online format. More neutral wording would have been preferable. We understand that the questions from the survey appear to be more directional to virtual meetings. However, the purpose of the survey was to understand committee members’ viewpoint on virtual rather than compared to face-to-face. -“Earlier proposals in the day received wider participation but by the end of the meeting, few people could contribute, understandable as it was a long day.” – this is likely the same in face-to-face meetings. - A description of the scoring process may be useful background. We have provided additional information about the scoring on page 15. - \"view an unconscious bias video prior to reviewing applications\" the NHMRC encourage reviewers to do the unconscious bias training (“Harvard Implicit Bias” https://www.nhmrc.gov.au/funding/peer-review). Thank you. We have included the NHMRC as an example of a funding organisation actively encouraging reviewers to do unconscious bias training. - \"some clustering of scores\" clustering within the scores from the same committee member (e.g., scores for feasibility, novelty) or across the committee? We have amended the clustering of scores sentence to provide greater clarity on this point. - Typo: \"Helsinkiv\". Thank you for your comments. We have addressed the typos accordingly and considered your additional comments in revising the manuscript." } ] }, { "id": "284400", "date": "21 Jun 2024", "name": "Carter Bloch", "expertise": [ "Reviewer Expertise Research funding", "research evaluation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper on a very relevant topic. It is important to understand how funding review processes work, in particular the use of online meetings to deliberate on proposals. The paper is well-written and clear. The paper is based on a mixed methods data collection which is very thoroughly described. The analysis is also very clear, where the paper brings out a number of interesting results, and utilizes a number of interview quotes and survey results to support the analysis. I have a number of more specific comments here:\nThe “results” part of the abstract describes the three main themes of the coding analysis, but I question whether these are really results. I think the paper has a number of more concrete findings, which could be summarized here.\nThe paper refers to whether the review processes are “effective, fair and transparent”. It would be helpful to spell out what is meant by these, particularly in the analysis where the paper concludes that online processes are “fair”. It would be good to elaborate on this (for example, in the Conclusion).\nFor the ethical process, you note that a Participant Information Sheet was sent out to informants prior to the data collection, but you also secured written consent from all informants? Typically it is either one or the other.\nOne aspect that I would have liked to see elaborated in greater detail is the issue of whether or how online meetings influence the scoring and selection process. Does it affect who receives the grants, and potentially also the diversity of projects (or other characteristics), or are there any indications of this? The overall conclusion appears to be that it does not affect the process, but the analysis hints at differences that could come into play in this regard. An example here could be in terms of how cases with the greatest difference in individual assessment are handled, or to what extent all committee member opinions are solicited and taken into consideration. Connected to this, it would be helpful to provide greater detail on the scoring process and how proposals are accepted.\nThe analysis is based on three data sources, observation, interviews and a survey. Based on my read of the method and data collection, I expected that the observation study would be at the centre of the analysis, supplemented by insights from the interviews and the survey. However, my impression of the results section is that it is the other way around. There are a few places where the analysis draws on the observations, but they are few and far between. It would be helpful to bring the observations more into play in the analysis, particularly concerning discussions and selection of projects.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11949", "date": "03 Jul 2024", "name": "Amanda Blatch-Jones", "role": "Author Response", "response": "The “results” part of the abstract describes the three main themes of the coding analysis, but I question whether these are really results. I think the paper has a number of more concrete findings, which could be summarized here. Thank you for your comment. Due to the word count it isn’t possible to add more to the results section, so we opted to include the identified themes along with the subthemes. The paper refers to whether the review processes are “effective, fair and transparent”. It would be helpful to spell out what is meant by these, particularly in the analysis where the paper concludes that online processes are “fair”. It would be good to elaborate on this (for example, in the Conclusion). Thank you for your comment. We have amended the introduction to take account of your comment. For the ethical process, you note that a Participant Information Sheet was sent out to informants prior to the data collection, but you also secured written consent from all informants? Typically it is either one or the other. The PIS was not used to secure participant consent, a separate process was used to gain consent from the participants. The PIS gave information about the study and why we were contacting them. Some participants completed the written consent form, but others did not. To be consistent and ensure that those who were interviewed were comfortable, the researcher read the consent form with the participants prior to conducting the interview (and gained verbal consent and give the participant the opportunity to ask questions prior to the interview). For the survey, written consent was requested prior to completing the survey. Written consent was not requested from the observations as consent was gained from the programme directors prior to the meeting and all members had a pre-emptive opt-out option two weeks prior to the meeting. One aspect that I would have liked to see elaborated in greater detail is the issue of whether or how online meetings influence the scoring and selection process. Does it affect who receives the grants, and potentially also the diversity of projects (or other characteristics), or are there any indications of this? The overall conclusion appears to be that it does not affect the process, but the analysis hints at differences that could come into play in this regard. An example here could be in terms of how cases with the greatest difference in individual assessment are handled, or to what extent all committee member opinions are solicited and taken into consideration. Connected to this, it would be helpful to provide greater detail on the scoring process and how proposals are accepted. Thank you for your comment. The scoring process did not differ to what was used in face-to-face meetings prior to COVID-19. Therefore, the main focus was not on the differences in the scoring but whether there were any practical challenges through doing this online. We have described some of these differences but we found that it did not affect the virtual process for decision-making. We have now added information about the scoring process, which also picks up your point about how proposals are accepted (e.g., proposals are ranked at the end to determine which applications meet the threshold). The analysis is based on three data sources, observation, interviews and a survey. Based on my read of the method and data collection, I expected that the observation study would be at the centre of the analysis, supplemented by insights from the interviews and the survey. However, my impression of the results section is that it is the other way around. There are a few places where the analysis draws on the observations, but they are few and far between. It would be helpful to bring the observations more into play in the analysis, particularly concerning discussions and selection of projects. We have tried to be consistent on drawing from all three data sources, given the richness of the data and length of the manuscript. The development of the themes and sub-themes were predominantly from the observations with the survey and interview data used to confirm what was observed from the perspective of the participants. The quotes used in the manuscript come mainly from the survey and interviews purely because conversations were not recorded verbatim during the observations. The quotes are used to add context to the data but the observations played a big role in the development of the themes. As directed by the research questions, we explored the role of virtual funding committees to inform decision-making, rather than focusing on how applications were selected. We feel that the discussions during the committee meetings are consistently covered in the results, which forms the basis of the themes." } ] }, { "id": "284407", "date": "27 Jun 2024", "name": "Becky Ioppolo", "expertise": [ "Reviewer Expertise research on research", "science policy", "evaluation", "science funding", "qualitative research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBlatch-Jones et al. investigate how the use of virtual platforms for funding committees in NIHR during a COVID-19 pandemic impacted the members of those committees, with implicit comparisons to face-to-face meetings, which were used previously. Through interviews, an online survey and observations of recorded meetings, the research team identified how committee members and funder staff experienced virtual meetings and the consequences the virtual format had on the meeting’s effectiveness and personal aspects. This is an interesting paper which is well written and easy to follow. I’m surprised there isn’t more research on the social / interpersonal dimension of funding committee members.\nThe introduction could have benefitted from more explicit context about the importance of work like this by making mentions of bias etc. to underscore the implications of human fallibility on decision making about scientific / “objective” concerns. When the people involved in the funding committees are not fully engaged or comfortable, it theoretically could lead to different funding outcomes, which is why this work is important.\nI felt there could have been better separation between considerations that are relevant to virtual versus face-to-face and considerations that are relevant in both virtual and face-to-face, especially in Table 2. Also, you mention “The amount and level of prior preparation for virtual committee meetings was frequently mentioned by NIHR staff and all eventualities were considered to try and prevent any delays during the actual committee meeting.” But how much does this level of preparation differ from face-to-face meetings? You had also mentioned that the voting software was used in both meeting formats, so what is the net effect of moving to virtual for NIHR staff?\nClarifications:\nWere/are the recordings publicly available or not? You say: “The virtual funding committees were recorded on the online platform used by NIHR staff to enable and assist in the minutes of the meeting outcomes which are made publicly available on the NIHR website.” And “The online funding committee meeting recordings were deleted once they were analysed.” It was unclear what your assumed response rate for the survey would be: “We aimed to receive a range of between 160 to 240 responses based on the average size of 20 committee members per research programme.“ Was this your aim or was this the maximum number of possible respondents? 20* how many programmes? Table 1: Will you explain what some of the barriers respondents considered might be? Or the scale of those barriers? Simply asking ‘are there barriers’ suggests that the answer is yes (because it is difficult to assert the complete absence of any barriers), but how intense are those barriers and what is the net effect with the positives? I felt that the authors could expand upon why or how virtual meetings are “less free flowing and more structured” or face-to-face meetings less so. You say it’s clear they are conducted differently but don’t explain why. In face-to-face meetings to people talk over each other more? Have more whispered asides between participants? Why isn’t/shouldn’t a face-to-face meeting be “structured”?\n\nTypos / quick fixes\nThe image resolution in Figure 1 and Figure 2 is poor making the text appear fuzzy and difficult to read. Either the authors have introduced a typo into the quote, “Some people do not like the longet silences and find it hard to cope with,” or the quote could benefit from a [sic].\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11950", "date": "09 Jul 2024", "name": "Amanda Blatch-Jones", "role": "Author Response", "response": "The introduction could have benefitted from more explicit context about the importance of work like this by making mentions of bias etc. to underscore the implications of human fallibility on decision making about scientific / “objective” concerns. When the people involved in the funding committees are not fully engaged or comfortable, it theoretically could lead to different funding outcomes, which is why this work is important. Thank you for your comment. Although we have mentioned bias from previous research in the introduction, we have also amended the last section under the virtual interaction section of the introduction to pick up on your point about bias. I felt there could have been better separation between considerations that are relevant to virtual versus face-to-face and considerations that are relevant in both virtual and face-to-face, especially in Table 2. Also, you mention “The amount and level of prior preparation for virtual committee meetings was frequently mentioned by NIHR staff and all eventualities were considered to try and prevent any delays during the actual committee meeting.” But how much does this level of preparation differ from face-to-face meetings? You had also mentioned that the voting software was used in both meeting formats, so what is the net effect of moving to virtual for NIHR staff? The objective of the study was not to compare face-to-face and virtual funding committees. As we had not observed face-to-face committees it would have been unfair to try and make comparisons without first observing face-to-face meetings. We have, where possible, through the survey, asked participants about their experience of face-to-face Vs virtual meetings to offer this viewpoint. Table 2 was particularly focused on considerations of virtual funding committees in the future rather than comparing to face-to-face for the reason stated above. However, this is certainly something for consideration in the future (comparing face-to-face to virtual committees). We have amended the text under ‘the importance of preparation for virtual committee meetings’ to take account of your comment. Although we were aware that committee members were used to the voting software through the interviews and survey, we did not observe face-to-face meetings. Therefore, it would be unfair to make a comparison based on virtual observations alone. However, we have added a sentence to reflect your comment. Were/are the recordings publicly available or not? You say: “The virtual funding committees were recorded on the online platform used by NIHR staff to enable and assist in the minutes of the meeting outcomes which are made publicly available on the NIHR website.” And “The online funding committee meeting recordings were deleted once they were analysed.” The recordings are not publicly available. The NIHR staff recorded the committee meetings to assist them in the minutes of the meeting, which are publicly available. For the research study, we gained ethical approval to have a copy of the recording for research purposes only. When we completed analyses of the observations, the recordings were deleted. It was unclear what your assumed response rate for the survey would be: “We aimed to receive a range of between 160 to 240 responses based on the average size of 20 committee members per research programme.“ Was this your aim or was this the maximum number of possible respondents? 20* how many programmes? Thank you for your comment. We have added “(a range of 8 to 12 research programmes included in the cohort).” to state the number of research programmes included in the cohort. Each funding committee can vary in size, therefore we took the average of 20 across the minimum and maximum number of committees. Table 1: Will you explain what some of the barriers respondents considered might be? Or the scale of those barriers? Simply asking ‘are there barriers’ suggests that the answer is yes (because it is difficult to assert the complete absence of any barriers), but how intense are those barriers and what is the net effect with the positives? The barriers highlighted in the survey are presented in the manuscript under the relevant themes (and under Table 1), complementing the observations and interview data, rather than reporting on the survey responses as a single data source to the study. I felt that the authors could expand upon why or how virtual meetings are “less free flowing and more structured” or face-to-face meetings less so. You say it’s clear they are conducted differently but don’t explain why. In face-to-face meetings to people talk over each other more? Have more whispered asides between participants? Why isn’t/shouldn’t a face-to-face meeting be “structured”? The focus of the study was not to compare to face-to-face as we did not observe any face-to-face meetings as part of the study (all committee meetings were virtual). The comment was made by an interviewee (and similar comments by others). It is possible that it felt more structured as there was no social element and time keeping due to not having run virtual committees before. We have amended the paragraph to accommodate your comment. Typos / quick fixes The image resolution in Figure 1 and Figure 2 is poor making the text appear fuzzy and difficult to read. Either the authors have introduced a typo into the quote, “Some people do not like the longet silences and find it hard to cope with,” or the quote could benefit from a [sic]. The images were provided to F1000 and we will look at the resolution to ensure it is if high quality (for both figures). We have amended figure 2 completely and have adjusted figure 1 resolution. Thank you. This is a typo error and we have corrected it." } ] } ]	1	https://f1000research.com/articles/13-338
https://f1000research.com/articles/13-452/v1	07 May 24	{ "type": "Study Protocol", "title": "Living systematic review and meta-analysis of plasma-concentrations of antipsychotic drugs in carriers and non-carriers of variant CYP450 genotypes: Living systematic review protocol", "authors": [ "Filip Milosavljević", "Stefan Leucht", "Stefan Leucht" ], "abstract": "Introduction Carriers of variant alleles of genes that encode liver CYP450 and UGT enzymes may experience abnormal plasma levels of antipsychotics and, consequently, worse efficacy or tolerability. Although pharmacogenomics is a rapidly developing field, current guidelines often rely on limited, underpowered evidence. We have previously demonstrated that meta-analysis is a viable strategy for overcoming this problem. Here, we propose a project that will expand our previous work and create a living systematic review and meta-analysis of drug plasma level differences between carriers and non-carriers of variant genotype-predicted phenotypes for every pharmacokinetic drug-gene interaction relevant to commonly used antipsychotic drugs.\n\nProtocol First, a baseline systematic review and meta-analysis will be conducted by searching for observational pharmacogenomics-pharmacokinetic studies. Data on dose-adjusted drug plasma levels will be extracted, and participants will be grouped based on their genotype for each drug-gene pair separately. Differences in plasma drug levels between different phenotypes will be compared using a random-effect ratio-of-means meta-analysis. The risk of bias will be assessed using ROBINS-I, and the certainty of evidence will be assessed using GRADE. Following the establishment of baseline results, the literature search will be re-run at least once every six months, and the baseline data will be updated and re-evaluated as new evidence is published. A freely available website will be designated to present up-to-date results and conclusions.\n\nDiscussion This systematic review will provide evidence-based results that are continuously updated with evidence as it emerges in the rapidly developing field of pharmacogenomics. These results may help psychiatrists in their decision-making, as clinicians are becoming increasingly aware of the patients’ genetic data as testing becomes more widespread and cheaper. In addition, the results may serve as a scientific basis for the development of evidence-based pharmacogenomics algorithms for personalized dosing of antipsychotics to mitigate potentially harmful drug-gene interactions.", "keywords": [ "Living systematic review", "Pharmacogenomics", "Antipsychotic drugs", "Schizophrenia", "Therapeutic drug monitoring", "CYP450" ], "content": "1. Introduction\n\nPharmacotherapy for schizophrenia today is challenging because of (I) slow development of new drugs,1 (II) treatment resistance,2 (III) frequent need for treatment regimen adjustments3 (IV) high relapse rates4 and (V) risk of unpleasant adverse drug reactions.4 In such a landscape, approaches such as pharmacogenomics hold great potential for maximizing the effectiveness of currently available psychiatric drugs. It is proposed that mutations in genes that code liver CYP450 (Cytochrome p450) and UGT (UDP-glycosyltransferase) enzymes can cause either too low or too high plasma levels of antipsychotic,5 leading to a lack of efficacy or a higher risk of adverse reactions in some patients.6 The precise extent of these pharmacokinetic drug-gene interactions must be determined to produce a pharmacogenomics-informed dosing algorithm that can mitigate these changes.\n\nMeta-analysis can be an effective tool for overcoming low statistical power, which is a common problem in many pharmacogenomics-pharmacokinetic studies.7 We will build on our previous work on precise quantification of pharmacokinetic drug-gene interactions of antipsychotics8 by analyzing more drug-gene interactions and utilizing a living systematic review approach.\n\nSince pharmacogenomics is a rapidly developing field and the level of evidence in pharmacogenomics is often unsatisfactory,5,7 continuously updated evidence-based data sources regarding pharmacokinetic drug-gene interactions could be very informative to clinicians, as patients’ genomic data are more commonly available to clinicians because commercial pharmacogenomics tools are becoming more frequently used and cheaper over time.\n\nOur hypothesis is that polymorphisms in genes encoding CYP450 and/or UGT enzymes are associated with significant changes in dose-corrected plasma concentrations of antipsychotic drugs.\n\nDrug-gene pairs will be formed for every relevant polymorphic gene associated with every relevant antipsychotic drug. For every drug gene pair, our aim is to determine, as precisely as possible, the magnitude of the difference in dose-corrected plasma drug concentrations between carriers and non-carriers of variant genotype-predicted phenotypes for the given gene, and the certainty of this evidence. Table 1 presents the PICO format for the research questions.\n\nBoth general case of the research question and an example of Risperidone-CYP2D6 drug-gene pair are given.\n\n\n\n1) Carrying CYP2D6 poor metabolizer phenotype (PM);\n\n2) Carrying CYP2D6 intermediate metabolizer phenotype (IM);\n\n3) Carrying CYP2D6 ultra-rapid metabolizer phenotype (UM).\n\n\n2. Protocol\n\nThis protocol was written in accordance with the PRISMA-P (Preferred reporting items for systematic review and meta-analysis protocols) guideline.9 A checklist is presented in the extended data. The final report of this living systematic review will be written in accordance with MOOSE (Meta-analysis of observational studies) reporting guideline.10 This protocol is also registered with PROSPERO (CRD42024485626).\n\nThe following databases will be searched: ClinicalTrials.gov, Cochrane Library (Cochrane Database of Systematic Reviews=CDSR and Cochrane Central Register of Controlled Trials=CENTRAL), Embase, MEDLINE, PsycINFO and WHO ICTRP. No studies that were published prior to 1.1.1995. will be searched because of the extremely limited availability of PCR genotyping technology. Both published and unpublished clinical studies will be searched, without language restrictions. The search will be re-run every time the resulting paper is submitted for peer review, as well as continuously once every 6 months during the living meta-analysis maintenance.\n\nSearch strategy draft is presented in the extended data.\n\nBased on the information from CPIC,11 PharmVar12 and PharmGKB13 resources, drug-gene pairs of interest were identified and are presented in Table 2. If new relevant data emerge during the maintenance of this living meta-analysis, this list will be updated, and a new drug-gene pair will be included.\n\nDrug-gene pairs that will be considered for analysis are formed by pairing most clinically relevant antipsychotic drugs and liver enzymes that are coded by polymorphic genes and are substantially involved in the metabolism of said drugs.\n\nAmisulpride will not be analyzed, as it is mainly extracted unchanged via the kidneys. Lurasidone will not be analyzed because of the uncertain importance of drug-blood level measurements.6 Ziprasidone will not be analyzed because of its low inter-individual variability in plasma drug levels.14 First-generation antipsychotic drugs, other than chlorpromazine, perphenazine, haloperidol, and zuclopentixol, were not included because of their limited clinical utility today.\n\nFor every drug-gene pair of interest, the exposure group(s) (i.e., variant group; experimental group) and control group (i.e., wild-type group; reference group) will be defined based on the genotype-predicted phenotypes for the gene in question. In the cases of CYP2C19 and CYP2D6, genotype-predicted phenotypes are also called “Metabolizer groups” where “Normal” metabolizer group is considered as a control, and “Intermediate,” “Poor” and “Ultra-rapid” metabolizer groups are considered as 3 possible exposure groups. Definitions of the variant genotypes were adopted from consensus guidelines for every individual gene.11–13 Metabolizer groups defined by phenotyping with a probe drug will not be considered valid for the purpose of this review. Detailed definitions of the control and experimental groups based on CYP2D6, CYP3A4/5, CYP2C19, CYP1A2, UGT1A4 and UGT2B7 genotypes are presented in the extended data.\n\nPharmacokinetic parameters used to represent the drug plasma level will be included only if they are dose adjusted or if the entire cohort is administered the same dose. If necessary and possible, dose-adjusted pharmacokinetic parameters will be manually estimated by dividing the mean drug plasma level and mean drug dose using the Taylor expansion method.15 The potential impact of this estimation will be explored in a sensitivity analysis, where manually estimated dose-adjusted data will be excluded. In addition, for this calculation, a correlation between drug dose and drug plasma levels of ρ=0.5 will be assumed, and two alternative values, ρ=0.2 and ρ=0.8 will be used instead in the sensitivity analysis.\n\nDrug plasma pharmacokinetic parameters are often presented as body-weight-adjusted. To increase the scope of the review, body-weight-adjusted and unadjusted data will be treated as interchangeable. If body-weight-adjusted and unadjusted data are presented simultaneously, body-weight-adjusted data will be prioritized. This decision will be tested using sensitivity analysis, in which only body-weight-adjusted data will be included.\n\nPharmacokinetic parameters that will be used to assess drug plasma levels will be:\n\nI). Dose-adjusted steady-state drug plasma concentration (Css/D): Measured after five half-elimination times have elapsed since drug initiation or therapy regimen change, during which patients took stable doses of the drug with good compliance. Ideally, measurements were taken in the morning, just before the new dose was taken.\n\nII). Dose-adjusted area under the steady-state plasma concentration-time curve (AUC/D): Measured after five half-elimination times had elapsed since drug initiation or therapy regimen change, during which patients took stable doses of the drug with good compliance. Measurements were taken during the same period for all participants.\n\nIII). Single-dose plasma concentration (C) or area under the plasma concentration curve (AUC): Even though drug plasma level measurements are not usually considered valid before the steady-state has been achieved owing to unpredictable variability, single-dose pharmacokinetic studies will also be included owing to their high level of confounding control and strict study design. These studies often use homogeneous study groups of healthy volunteers with controlled intake of food, water, and other substances and drugs, with standardized blood sampling times. Thus, well-controlled single-dose pharmacokinetic parameters will be used interchangeably with steady-state parameters, and this decision will be tested in a sensitivity analysis where single-dose studies are excluded.\n\nIV). Reciprocal value of the total drug clearance (1/Cltotal): For drugs with a linear pharmacokinetic profile, the reciprocal value of the total clearance is equal to the AUC/D metric. If clearance is presented as median with interquartile range and/or minimum and maximum values, these values will be transformed into their reciprocal values, which will be further transformed into mean and standard deviation.16 This decision will be tested in the sensitivity analysis, where reciprocal value data are excluded.\n\nIf the blood sample for the Css/D measurement was not taken in the morning before the next dose, but the blood sampling time was consistent in the entire cohort, the study will be included; however, this decision will be tested in the sensitivity analysis where only studies with the ideal sampling times are included.\n\nIf the drug possesses one or more active metabolites that are present in the plasma in non-negligible amounts11 compared to the parent drugs, drug plasma levels will be expressed as “Active moiety,” i.e. the sum of the plasma parent drug levels and the plasma active metabolite levels.\n\nIf multiple pharmacokinetic parameters are present within the same study for the same cohort, Css/D will be prioritized because it is the most common readout.8 If the same pharmacokinetic parameter is presented for multiple time-points, the longest time point will be selected because of the lower chance that some of the included participants have not yet reached steady-state blood drug levels.\n\n\n\nI). For every drug-gene pair of interest, the study will be included if it reports drug plasma levels for control genotype-predicted phenotype and at least one variant genotype-predicted phenotype for the given gene.\n\nII). Studies involving participants of all ethnicities will be included. However, since the genotype frequencies vary drastically based on ethnic background, subgroup analysis will be performed to compare the results between European, East Asian, South Asian, African, and Admixed American origin cohorts.17\n\nIII). Studies in which the participants were treated with antipsychotic drugs were included. This includes studies on patients with schizophrenia, other psychiatric illnesses, and healthy volunteers. To assess the effect of this decision, studies on healthy volunteers will be excluded as a part of the sensitivity analysis.\n\nIV). All age groups were eligible for inclusion in principle, but the main results will be presented only for adults between 18 and 65 years of age. Results on participants of all ages, results only in adolescents (12-18 years old), and the results only for the elderly (>65 years old) will be explored in the sensitivity analysis.\n\nV). Studies will be included regardless of whether the participants were on a stable dosing regimen on enrolment, started drug regimen on enrolment, or switched to a new drug on enrolment.\n\nVI). Studies on both responders and treatment resistant patients will be included.\n\nVII). Studies on participants not exposed to co-medications, foods, or smoking, in cases where these factors are known to interfere with the given antipsychotic drug, are preferred because of the lower risk of confounding. Still, studies where participants are exposed to these confounders will be eligible for inclusion only if these factors are well balanced across different phenotype groups or corrected by the authors during data analysis. In addition, studies with co-medication/food/smoking confounding factors will be excluded as a part of the sensitivity analysis.\n\nVIII). Studies on participants taking any drug formulation (immediate release tablets, extended release tablets, long-acting injections, intravenous administration, etc.) will be eligible, and the data for all formulations will be used interchangeably in the main meta-analysis. To test this decision, every formulation that is not an immediate release tablet will be excluded as a part of the sensitivity analysis.\n\nIX). Prospective studies will be preferred because of the higher reliability of the pre-planned experiments.18 Nevertheless, retrospective studies are very common in pharmacogenomics and will be included to increase the scope of this review. To test this decision, retrospective studies will be excluded as a part of the sensitivity analysis.\n\nX). To increase the scope of this living systematic review, both published and unpublished studies will be included.\n\n\n\nI). Studies on pregnant women will be excluded due to unpredictable pharmacokinetics during pregnancy.\n\nII). Studies with a critical overall risk-of-bias grade based on the ROBINS-I (Risk of bias in non-randomised studies of interventions) tool19 will be excluded (four possible risk-of-bias grades in the ROBINS-I tool are low, moderate, serious, and critical risk of bias, ranked from the best to the worst grade).\n\nIII). Studies with a substantial proportion of the population consisting of non-compliers will be excluded because of the risk of erroneous drug plasma level measurements.\n\nIV). Studies on participants with severe kidney or liver impairments or other severe conditions that interfere with drug pharmacokinetics (Malabsorption, Hypoproteinemia, etc.) will be excluded because of unpredictable changes in the pharmacokinetics of these participants.\n\nV). Studies with unbalanced known confounding factors (co-medications, food, cigarette smoke) for drug plasma levels between normal and variant genotype-predicted phenotype groups will be excluded.\n\nVI). Population pharmacokinetic studies will be excluded due to incompatibility with other included studies.\n\nBoth the screening and selection of the results of the systematic search will be performed independently by two researchers, and any disputes will be resolved by consensus with a third researcher.\n\nScreening will be performed based on the titles and abstracts of the search results. During the screening, search results will be labels as “not relevant,” “relevant” or “unclear relevance.”\n\nFor the search results labelled as “relevant” and “unclear relevance,” full text retrieval will be attempted. If the full text could not be retrieved, the corresponding author will be contacted to provide the full text. Next, the retrieved full texts will be assessed for eligibility based on the inclusion and exclusion criteria.\n\nProcess of screening and selection will be visually reported using PRISMA 2020 flowchart.20\n\nData will be extracted using an internally developed standardized form.\n\nMeans and standard deviations of drug plasma levels, as well as the number of participants, will be extracted. Means and standard deviations will be estimated using standard procedures if the data are presented as geometric mean and 95%CI,21 as median, interquartile range, minimum, and maximum16 or as mean values and the p-value of the mean difference.21 If the Plasma levels are presented graphically but not numerically, numerical data will be extracted from the digital image of the given graph.22\n\nIf patients were not grouped into genotype-predicted phenotypes according to our desired criteria, manual regrouping will be performed when possible. For example, if plasma levels in participants carrying CYP2D61/10 and 1/5 are presented separately, but there are no data for CYP2D6 IM collectively, the mean plasma levels in 1/10 and 1/5 carriers will be summed manually to form the mean IM plasma levels.21\n\nRegarding demographics, data on ethnicity, sex, and age distribution will be collected. Regarding clinical characteristics, data on the participants’ diagnosis, drug dose, body weight, duration of treatment, blood sampling time, co-medications, and other drug-specific confounders will be collected. Finally, data on study design and the characteristics of reported pharmacokinetic parameters will be collected.\n\nIf cohorts of two or more clinical studies overlap, inclusion of the studies will be performed to maximize the inclusion of as many unique participants as possible while not having any repeating participants.\n\nIf the included manuscript suggests that the data of interest were measured but not presented, the corresponding author will be contacted to provide the data. If this is not possible, the study will be labelled as “Awaiting assessment”. Studies with missing data will be reported as a list and considered during the interpretation of publication bias using funnel plots.\n\nThe ROBINS-I tool19 will be used to assess the seven domains of bias by two researchers independently. Disputes will be resolved by consensus with a third researcher. Based on the semi-quantitative grades for all seven domains, the overall grade for each study will be assigned. Due to the nature of observational studies that will be searched, it is expected for a small number of included studies, if any, to receive the best grade: “Low” risk-of-bias. Instead, it is expected that studies with relatively better design to be graded as having “Moderate” risk-of-bias, while the studies with relatively worse design to receive “Serious” of “Critical” risk-of-bias grade. “No information” overall risk-of-bias grade will be used sparingly, i.e. everywhere where reasonable assumptions regarding the signalling question cannot be made. Studies with a risk of bias grade other than “moderate” or “low” will be excluded as a part of the sensitivity analysis.\n\nCertainty of evidence will be assessed using the GRADE tool23 across five domains of evidence certainty; overall certainty of evidence will also be assessed for every drug-gene pair of interest. GRADE scoring will be performed independently by two researchers, and disputes will be resolved by consensus with a third researcher.\n\nQuantitative data needed for the meta-analysis includes the mean plasma level, standard deviation, and number of participants in the given study group. For every drug-gene pair analyzed, all experimental groups will be compared pairwise with their corresponding control group. Drug plasma levels will be compared between the groups using the ratio-of-means (RoM) effect measure24 and its 95% confidence interval. This will allow for an intuitive interpretation of the results, as well as the comparison of different pharmacokinetic parameters of drug plasma levels within one analysis. Our previous research showed a high probability of high heterogeneity within the included studies; therefore, a random-effect meta-analysis will be performed. Specifically, RoM=1.15 will be interpreted as 15% higher, and RoM=0.85, which will be interpreted as a 15% lower drug plasma level in the experimental group compared to the control group. RoM values for individual values will be log-transformed and pooled using the inverse-variance method to produce the grand-mean.\n\nAs a secondary analysis, all meta-analysis results calculated as RoM will also be calculated as the Standardized Mean Difference (SMD, Hedges’ g). This is aimed at providing an alternative interpretation of the results, which is important considering the rare use of the RoM effect measure.\n\nHeterogeneity will be assessed using the I2 metric, where I2>50% will be considered substantial heterogeneity.21 In the case of substantial heterogeneity, the effect of outlier results on the overall results will be assessed using a sensitivity analysis. Further, heterogeneity will be explored using other sensitivity and subgroup analyses (previously described) to explore what clinical or demographic factors may be the cause of study misalignment.\n\nPotential publication bias and small-study effects will be assessed using contour-enhanced funnel plots25 and Egger’s test.26 If fewer than 10 studies are included for a certain drug-gene pair, publication bias will not be performed, as recommended.25 The critical p-value for Egger’s test will be set at p<0.10, as suggested.26\n\nSensitivity analysis will be used to test the robustness of the obtained results with regard to changes to the inclusion/exclusion criteria. The following sensitivity analyses will be performed: 1) Exclusion of the manually dose-adjustment plasma level data; 2) Usage of ρ=0.2 and ρ=0.8 as an assumed correlations levels between drug dose and drug plasma levels for manual dose-adjustment of plasma levels; 3) Exclusion of body-weight-unadjusted data; 4) Exclusion of the data estimated from reciprocal clearance values; 5) Exclusion of studies with suboptimal time of plasma sampling; 6) Inclusion of data on non-adults; 7) Exclusion of the studies with suboptimal confounding control; 8) Exclusion of “Single drug dose” studies; 9) Exclusion of the studies with “Serious” risk-of-bias overall score; 10) Exclusion of healthy volunteer data; 11) Exclusion of retrospective studies and 12) Exclusion of the data on drug formulations other than immediate release tablets.\n\nSubgroup analyses will be used to compare the meta-analysis results only between participants of different ethnic backgrounds.\n\nFollowing an initial literature search and data analysis, the baseline results and conclusions of the living systematic review and meta-analysis will be established. As these results are being published, designated website will be created to display these results, and thus the maintenance phase of the living systematic review will begin. In this phase, once every six months, a systematic literature search will be repeated, and two designated researchers will be tasked with screening and testing for eligibility of the new search results. Alternatively, if a sudden increase in the number of new publications is detected during the first year of maintenance, updates from that point onwards will be performed more often once every three months. The data from the eligible studies will be extracted in the same manner as during the establishment of the baseline results and will be added to the database of the baseline results. Updated results will then be published on the website, together with the change log. If a new discovery regarding pharmacogenomics of liver enzymes involved in the metabolism of antipsychotics emerges, the entire database will be updated accordingly in the shortest possible notice. This includes newly detected drug-gene pairs of interest and new consensus for genotype-determined phenotype classification.\n\nFor pragmatic reasons, once all analyzed drug–gene pairs either achieve satisfactory strength of evidence or experience no new publications for an extended period of time, updates of the living systematic review will be performed less frequently, and the update frequency will depend on the probability of new evidence emerging in the future.\n\nAs previously mentioned, the baseline results will be published in separate papers, while the up-to-date results and conclusions will be presented on a designated website. Papers published in scientific journals are planned from time to time during the living systematic review maintenance phase.\n\nAt the time of writing the manuscript (29. January 2024), the literature search strategy was designed, and a systematic search was not performed for either of the drug-genes of interest. No study screening, selection, or data extraction was performed at the time of writing.\n\n\n3. Discussion\n\nThis project has the potential to provide much-needed clinical information to the psychiatry community. Psychiatrists are becoming increasingly aware of the CYP450 genetic status of their patients, and the implementation of evidence-based knowledge of drug-gene interactions in psychiatrists’ decision-making will improve the quality of pharmacotherapy in psychiatry. This interaction will be facilitated by up-to-date reporting of the systematic review results on a designated website, which will be freely available to any interested clinician.\n\nFurthermore, several attempts have been made to create and test clinical tools for pharmacogenomics-guided pharmacotherapy for schizophrenia.27–29 Such tools aim to detect and mitigate potentially dangerous drug-gene interactions, and thus improve the efficacy and/or safety of antipsychotic drugs. One common approach is to completely avoid drugs known to interact negatively with the genetic makeup of a patient. This approach is criticized as potentially dangerous because it can overly restrict the list of effective options available to the patient.30 Another promising approach is to use genetic information to guide the dosing regimen of antipsychotic drugs, but due to limited and often conflicting evidence, genotype-guided dose regimen adjustments are often arbitrary. The results of this systematic review may facilitate the development of new and the improvement of existing pharmacogenomic tools by providing precise estimates of expected blood drug concentration changes in carriers of different genetic variants. This may help in the transition from arbitrary to evidence-based algorithms for the mitigation of potentially harmful drug-gene interactions related to the CYP450 and UGT genotypes.\n\nOne of the most substantial limitations of this project was the expected high risk of bias in the included studies. This is because of the very common naturalistic approach with insufficient confounder control in the eligible studies observed in our previous work.8 In addition, carriers of variant CYP450 or UGT alleles may be more likely to drop out due to their atypical drug plasma levels during observational studies, potentially biasing the results. Finally, there is a risk that some of the results of this systematic review may become outdated with the discovery of new alleles for some genes of interest. This could cause participants to become misclassified in the studies published prior to this discovery without the option to re-classify them post hoc, as this would require repeated genetic testing.\n\nEthical approval and written informed consent were not required.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare, LivingCYP - Living CYP - Extended data - F1000 25mar2024.docx, https://doi.org/10.6084/m9.figshare.25109675.v2. 31\n\nFigshare: PRISMA –P checklist for Living systematic review and meta-analysis of plasma-concentrations of antipsychotic drugs in carriers and non-carriers of variant CYP450 genotypes: Living systematic review protocol, https://doi.org/10.6084/m9.figshare.25109675.v2. 31\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nMiller AH, Raison CL: Burning down the house: reinventing drug discovery in psychiatry for the development of targeted therapies. Mol. Psychiatry. Jan 2023; 28(1): 68–75. PubMed Abstract \| Publisher Full Text\n\nHowes OD, Thase ME, Pillinger T: Treatment resistance in psychiatry: state of the art and new directions. Mol. Psychiatry. Jan 2022; 27(1): 58–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDold M, Leucht S: Pharmacotherapy of treatment-resistant schizophrenia: a clinical perspective. Evid. Based Ment. Health. May 2014; 17(2): 33–37. PubMed Abstract \| Publisher Full Text\n\nKaar SJ, Natesan S, McCutcheon R, et al.: Antipsychotics: Mechanisms underlying clinical response and side-effects and novel treatment approaches based on pathophysiology. Neuropharmacology. Aug 1 2020; 172: 107704. PubMed Abstract \| Publisher Full Text\n\nJukic M, Milosavljevic F, Molden E, et al.: Pharmacogenomics in treatment of depression and psychosis: an update. Trends Pharmacol. Sci. Dec 2022; 43(12): 1055–1069. PubMed Abstract \| Publisher Full Text\n\nMauri MC, Paletta S, Di Pace C, et al.: Clinical Pharmacokinetics of Atypical Antipsychotics: An Update. Clin. Pharmacokinet. Dec 2018; 57(12): 1493–1528. PubMed Abstract \| Publisher Full Text\n\nShekhani R, Steinacher L, Swen JJ, et al.: Evaluation of Current Regulation and Guidelines of Pharmacogenomic Drug Labels: Opportunities for Improvements. Clin. Pharmacol. Ther. May 2020; 107(5): 1240–1255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilosavljevic F, Bukvic N, Pavlovic Z, et al.: Association of CYP2C19 and CYP2D6 Poor and Intermediate Metabolizer Status With Antidepressant and Antipsychotic Exposure: A Systematic Review and Meta-analysis. JAMA Psychiatry. Mar 1 2021; 78(3): 270–280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. Jan 2 2015; 349: g7647. PubMed Abstract \| Publisher Full Text\n\nStroup DF, Berlin JA, Morton SC, et al.: Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA. Apr 19 2000; 283(15): 2008–2012. PubMed Abstract \| Publisher Full Text\n\nHiemke C, Bergemann N, Clement HW, et al.: Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology: Update 2017. Pharmacopsychiatry. Jan 2018; 51(1-02): 9–62. PubMed Abstract \| Publisher Full Text\n\nGaedigk A, Casey ST, Whirl-Carrillo M, et al.: Pharmacogene Variation Consortium: A Global Resource and Repository for Pharmacogene Variation. Clin. Pharmacol. Ther. Sep 2021; 110(3): 542–545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhirl-Carrillo M, Huddart R, Gong L, et al.: An Evidence-Based Framework for Evaluating Pharmacogenomics Knowledge for Personalized Medicine. Clin. Pharmacol. Ther. Sep 2021; 110(3): 563–572. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeedham C, Miceli JJ, Obach RS: Ziprasidone metabolism, aldehyde oxidase, and clinical implications. J. Clin. Psychopharmacol. Jun 2003; 23(3): 229–232. PubMed Abstract \| Publisher Full Text\n\nStuart A, Ord JK: Kendall’s Advanced Theory of Statistics. 6th ed.Arnold; 1998; vol. 1. .\n\nWan X, Wang W, Liu J, et al.: Estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range. BMC Med. Res. Methodol. Dec 19 2014; 14: 135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou Y, Ingelman-Sundberg M, Lauschke VM: Worldwide Distribution of Cytochrome P450 Alleles: A Meta-analysis of Population-scale Sequencing Projects. Clin. Pharmacol. Ther. Oct 2017; 102(4): 688–700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNorvell DC: Study types and bias-Don’t judge a study by the abstract’s conclusion alone. Evid. Based Spine Care J. Aug 2010; 1(2): 7–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSterne JA, Hernan MA, Reeves BC, et al.: ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ. Oct 12 2016; 355: i4919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. Mar 29 2021; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNasser M: Cochrane Handbook for Systematic Reviews of Interventions. Am. J. Public Health. Jun 2020; 110(6): 753–754. Publisher Full Text\n\nRohatgi A: WebPlotDigitizer 4.6.Reference Source2022;\n\nSchünemann H, Brozek J, Guyatt G, et al.: GRADE Handbook. UK: Cochrane collaboration London; 2013.\n\nFriedrich JO, Adhikari NKJ, Beyene J: The ratio of means method as an alternative to mean differences for analyzing continuous outcome variables in meta-analysis: A simulation study. BMC Med. Res. Methodol. May 21 2008; 8: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSterne JA, Sutton AJ, Ioannidis JP, et al.: Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ. Jul 22 2011; 343: d4002. PubMed Abstract \| Publisher Full Text\n\nEgger M, Davey Smith G, Schneider M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. Sep 13 1997; 315(7109): 629–634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarrascal-Laso L, Franco-Martín MA, García-Berrocal MB, et al.: Application of a Pharmacogenetics-Based Precision Medicine Model (5SPM) to Psychotic Patients That Presented Poor Response to Neuroleptic Therapy. J. Pers. Med. Dec 2020; 10(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJurgens G, Andersen SE, Rasmussen HB, et al.: Effect of Routine Cytochrome P450 2D6 and 2C19 Genotyping on Antipsychotic Drug Persistence in Patients With Schizophrenia: A Randomized Clinical Trial. JAMA Netw. Open. Dec 1 2020; 3(12): e2027909. PubMed Abstract \| Publisher Full Text\n\nKang ZW, Qin Y, Sun YT, et al.: Multigenetic Pharmacogenomics-Guided Treatment vs Treatment As Usual Among Hospitalized Men With Schizophrenia. JAMA Netw. Open. Oct 6 2023; 6(10): e2335518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoldberg JF, Rosenblat JD, McIntyre RS, et al.: Letter to the Editor: Clinical versus statistical significance of pharmacogenomic-guided antidepressant therapy: What’s really being measured and marketed?. J. Psychiatr. Res. Jul 2019; 114: 208–209. PubMed Abstract \| Publisher Full Text\n\nMilosavljević F: Living CYP - Extended data - F1000 25mar2024.docx. figshare. 2024. Publisher Full Text" }	[ { "id": "275387", "date": "31 May 2024", "name": "Jhohann Richard de Lima Benzi", "expertise": [ "Reviewer Expertise Clinical pharmacology", "pharmacokinetics", "pharmacometrics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript under review presents a comprehensive systematic review protocol into the relevance of CYP pharmacogenetics in antipsychotic (AP) therapy, offering a clear and well-written discourse on the topic. The study's goal is undeniably pertinent, addressing a critical aspect of personalized medicine in psychiatric care. I commend the authors for their meticulous approach and the significance of their endeavors. However, I would like to suggest some considerations to enrich the manuscript.\nFirstly, the inclusion or mention of the importance of drug transporters in AP therapy warrants attention, particularly those expressed in the blood-brain barrier. Given their pivotal role in drug disposition and response, their omission from the review raises questions about their relevance in AP pharmacotherapy. Exploring the available pharmacogenetic data pertaining to these transporters could provide valuable insights into their impact on treatment outcomes.\nSecondly, the decision to exclude data based on probe phenotyping drug data when defining metabolizer groups requires clarification. Elaborating on the rationale behind this exclusion would enhance the transparency and reproducibility of the study, ensuring a more robust analysis of CYP pharmacogenetics in AP therapy.\nThirdly, I respectfully disagree with the assertion that single-dose pharmacokinetic parameters can be used interchangeably with steady-state parameters, particularly in the context of AP drugs with non-linear pharmacokinetics. Given the complexities inherent in non-linear PK profiles, a sensitivity analysis excluding single-dose studies may not adequately capture the dynamic nature of drug metabolism. It is imperative to devise alternative strategies to handle such data, perhaps through modeling techniques or subgroup analyses, to ensure the integrity and reliability of the findings.\nIn conclusion, while the manuscript admirably addresses the relevance of CYP pharmacogenetics in AP therapy, the incorporation of drug transporter considerations, clarification on data exclusion criteria, and careful handling of non-linear PK data are essential for a more comprehensive and nuanced analysis.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11941", "date": "09 Jul 2024", "name": "Filip Milosavljević", "role": "Author Response", "response": "\"The manuscript under review presents a comprehensive systematic review protocol into the relevance of CYP pharmacogenetics in antipsychotic (AP) therapy, offering a clear and well-written discourse on the topic. The study's goal is undeniably pertinent, addressing a critical aspect of personalized medicine in psychiatric care. I commend the authors for their meticulous approach and the significance of their endeavors. However, I would like to suggest some considerations to enrich the manuscript.\" We thank the reviewer for positive feedback. \"Firstly, the inclusion or mention of the importance of drug transporters in AP therapy warrants attention, particularly those expressed in the blood-brain barrier. Given their pivotal role in drug disposition and response, their omission from the review raises questions about their relevance in AP pharmacotherapy. Exploring the available pharmacogenetic data pertaining to these transporters could provide valuable insights into their impact on treatment outcomes.\" Indeed, drug efflux pumps are coded by highly polymorphous genes (ABCB1, ABCG2, ABCC2…) and the research of their pharmacogenomics has been very active in the recent years. We initially omitted to mention drug transporter in an effort to keep our report concise, but we agree with the reviewer that these genes have to be addressed in order to provide comprehensive overview of pharmacogenomics of antipsychotic drug pharmacokinetics, and that the omission of drug transporter genes from our analysis has to be justified in the manuscript. The main reason to exnclude pharmacogenomic studies on efflux pumps from our review is potential inappropriateness of our main outcome to capture the impact of pharmacogenomics of drug transporters on antipsychotic therapy. Our review is focused on plasma levels of antipsychotics, and in our analysis we will assume that cerebrospinal-fluid-to-plasma ratios of antipsychotic drugs are similar between people with different CYP450/UGT genotypes. Since drugs transporters are expressed on blood-brain barrier, in some antipsychotic drugs it has been demonstrated that mutations of drug transporter genes such as ABCB1 can change cerebrospinal-fluid-to-plasma ratios [PMID: 21148022, PMID: 20599439, PMID: 24666334]. This means that, for example, if two groups of patients with different ABCB1 mutations have the same antipsychotic plasma levels, they may not have the same antipsychotic cerebrospinal fluid levels, which puts into question the appropriateness of drug plasma level as a biomarker. Therefore, we believe that analyzing drug efficacy and safety directly, or analyzing cerebrospinal fluid drug levels are more appropriate outcomes for the analysis of drug transporters pharmacogenomics, all of which are out of the scope of this review. Introduction section now includes short summary of the potential significance of efflux pumps, and Section 2.2. now includes the justification of our justification not to include efflux pump pharmacogenomic data. \"Secondly, the decision to exclude data based on probe phenotyping drug data when defining metabolizer groups requires clarification. Elaborating on the rationale behind this exclusion would enhance the transparency and reproducibility of the study, ensuring a more robust analysis of CYP pharmacogenetics in AP therapy.\" We agree with the reviewer that this decision needs more clarification. Also, upon revision of our manuscript, we noticed that our previous wording “for the purpose of this review, phenotyping will not be considered valid” can be interpreted as us questioning the validity of phenotyping method, which is not the case. Instead, section 2.3 is now updated to introduce pros and cons of both phenotyping and genotyping approaches and the justification why we opted not to use them interchangeably in this review. \"Thirdly, I respectfully disagree with the assertion that single-dose pharmacokinetic parameters can be used interchangeably with steady-state parameters, particularly in the context of AP drugs with non-linear pharmacokinetics. Given the complexities inherent in non-linear PK profiles, a sensitivity analysis excluding single-dose studies may not adequately capture the dynamic nature of drug metabolism. It is imperative to devise alternative strategies to handle such data, perhaps through modeling techniques or subgroup analyses, to ensure the integrity and reliability of the findings.\" We thank the reviewer for raising this concern. Even though the majority of antipsychotics have linear pharmacokinetic profile, sublingual asenapine, oral lumateperone, oral chlorpromazine and oral perphenazine possess non-linear pharmacokinetic profiles (PMID: 29915922, PMID: 32060882, PMID: 8157044, PMID: 3933033). We agree with the reviewer that for the given examples, data from single dose studies and steady state studies cannot be used interchangeably and we apologize for our omission. As we are currently not aware of any model published in the literature designed to predict steady state levels of given four drugs based on single-dose data, we will, for the time being, opt to exclude single-dose studies for asenapine, lumateperone, chlorpromazine and perphenazine. We expect very small (if any) impact of this decision on the results of our meta-analyses, as we, based on our previous experience, expect to detect very few pharamcogenomic studies in the literature on these 4 drugs. If instead we happen to detect many eligible single-dose studies for these 4 drugs, we will post-hoc find appropriate model developed for other drugs that may fit the drug in question. This clarification is now reflected in the manuscript. \"In conclusion, while the manuscript admirably addresses the relevance of CYP pharmacogenetics in AP therapy, the incorporation of drug transporter considerations, clarification on data exclusion criteria, and careful handling of non-linear PK data are essential for a more comprehensive and nuanced analysis.\" We thank the reviewer again for the constructive criticism that will improve the quality of this paper. We have no further comments." } ] }, { "id": "280401", "date": "06 Jun 2024", "name": "Pierre Baumann", "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\nGeneral comment:\nThe authors propose a protocol for future studies on the relationship between drug plasma concentrations in patients treated with antipsychotics and their pharmacogenetic status regarding genetic polymorphisms of cytochrome P-450 isoforms and/or UGT enzymes (cf Table 1, criterion O (outcome)). It would have been of interest to include a part which concerns the clinical outcome (efficacy, effectiveness, adverse effects) of the patients, as the present protocol will not allow to clearly show the clinical relevance of pharmacokinetic-pharmacogenetic exams. However, the present project is already ambitious and the inclusion of a regularly occurring update is highly promising and novel.\nOther comments and questions:\nThe issues dealing with pharmacokinetics and pharmacogenomics presented in the Introduction should be presented in more details as some references are not adequate. Indeed, some citations lack precision, e.g. ref 5 and 6, in the sentence: “It is proposed that mutations in genes that code liver CYP450 (Cytochrome p450) and UGT (UDP-glycosyltransferase) enzymes can cause either too low or too high plasma levels of antipsychotic,5 leading to a lack of efficacy or a higher risk of adverse reactions in some patients.6 The citation of reference 5 suggests that it presents data about the importance of UGT in the pharmacogenomics of antipsychotics. However, UGT is only mentioned as a key word (and only as UGT-2B.. but not as UGT-1A..). In ref 6, UGT is not even mentioned. Therefore, the reader with limited knowledge in this field may not get a full picture of the importance of the subject. In summary, the Introduction should be extended!\nTable 1 presents in “Intervention I” the different phenotypes using the example risperidone. The reader is then supposed to presume that this presentation is also valid for UGT enzymes (eg gene-drug pair: UGT-olanzapine), but this classification (poor, intermediate, extensive, ultrarapid) is seemingly not introduced for these polymorphisms. If however this is the case, this should be presented in some details and corresponding references should be presented.\nIndeed, the average reader will not be familiar with the genetic polymorphisms of conjugating enzymes, and in particular, with their relevance for antipsychotics. In other words: are there PM, EM, UM, IM in patients treated with e.g. asenapine or olanzapine, with regard to the metabolism by UGT-enzymes? (Admittedly, in the suppl. material is some information, but the protocol should be more precise, especially in Introduction). It is a fact that among cytochrome P-450 isozymes, only about half a dozen isozymes play a role in the metabolism of antipsychotics. In contrast, there are considerably more isozymes which contribute to the conjugation of drugs, but their individual role in the metabolism of antipsychotics is poorly characterized.\nThe pioneering study of Haslemo et al. (CPT, 2012, 221) (besides that of Erickson et al., Pharmacogen Genom 2011, 539) shows that plasma concentrations of conjugated olanzapine (10-N-ola-glucuronide) differ in olanzapine treated patients classified according to their UGT1A genotypes, but the difference is negligible for (unconjugated) olanzapine concentrations. This rises the question whether in the present meta-analysis study both ola and conjugated ola concentrations will be considered. It could be then of interest to examine the relationships between the UGT genotypes and the ratios parent compound/metabolite (Ola/10-N-ola-glucoronide).\nThis approach may also be valuable for all other antipsychotics, whether they are metabolized by cytochrome P-450 or by polymorphic conjugating enzymes, or by both types (e.g. risp/9-OH-risp; 9-OH-risp/conjug. 9-OH-risp; etc). Probably of little importance for the establishment of the protocol, it is to mention that in the case of risperidone, its hydroxylation to 9-OH-risperidone is stereoselective, in that CYP2D6 preferentially forms (+)-9-OH-risperidone rather than (-)-9-OH-risperidone (Yasui-Furukori et al. Drug Metab. Disp 2001, 1263). It should be considered, as rarely, stereoselective procedures were used in the published studies, the net effect of the genotype may be masked when non-stereoselective procedures are used.\nThe authors exclude some antipsychotics, but the mentioned reasons are not always evident, and again, the citations do not always correspond to the statements. Therefore, the presentation of the reference list demands to be improved. Eg, 2.2. Drug gene pairs …: “Lurasidone will not be analyzed because of the uncertain importance of drug-blood level measurements.” Ref 6 is cited, but this ref does apparently not deal with this subject. Actually, lurasidone is mainly a substrate of CYP3A4/5. As its blood levels strongly depend on dietary conditions, this may be an important reason for recommending TDM (cf Preskorn 2013: Lurasidone should be administered with food-at least 350 kcal-to ensure maximum exposure). However, one can agree with the decision of the authors, as this drug belongs to the group of drugs mentioned in 2.5. VII (study inclusion citeria) resp. 2.6. V.\n\n2.4. Definition of the outcome As pointed out by the authors, weight corrected drug concentrations may be advantageous, but they are often not available.\nSteady-state plasma concentrations: In the case of risperidone, there may be a problem in that in extensive metabolizers (CYP2D6), its T1/2 = 2 - 4h, but in poor metabolizers, considerably longer, comparable to that of its metabolite 9-OH-risperidone. This means that in EM, risp. steady-state concentrations will hardly be reached in EM but only in PM.\nExtended data: March : 6. UGT1A4 “At the time of writing, there is no consensus regarding the classification of different UGT1A4 diplotypes into metabolizer groups. Therefore, pragmatically, alleles known to affect CYP1A2 activity with Minor allelic frequencies >1% in the world population will be considered for this analysis.” Not clear: this suggests that there is a relationship CYP1A2 – UGT1A4 (?).\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11942", "date": "09 Jul 2024", "name": "Filip Milosavljević", "role": "Author Response", "response": "\"The authors propose a protocol for future studies on the relationship between drug plasma concentrations in patients treated with antipsychotics and their pharmacogenetic status regarding genetic polymorphisms of cytochrome P-450 isoforms and/or UGT enzymes (cf Table 1, criterion O (outcome)). It would have been of interest to include a part which concerns the clinical outcome (efficacy, effectiveness, adverse effects) of the patients, as the present protocol will not allow to clearly show the clinical relevance of pharmacokinetic-pharmacogenetic exams. However, the present project is already ambitious and the inclusion of a regularly occurring update is highly promising and novel.\" We thank the reviewer for positive comment and for recognizing the scale of this project. \"Other comments and questions: The issues dealing with pharmacokinetics and pharmacogenomics presented in the Introduction should be presented in more details as some references are not adequate. Indeed, some citations lack precision, e.g. ref 5 and 6, in the sentence: “It is proposed that mutations in genes that code liver CYP450 (Cytochrome p450) and UGT (UDP-glycosyltransferase) enzymes can cause either too low or too high plasma levels of antipsychotic,5 leading to a lack of efficacy or a higher risk of adverse reactions in some patients.6 The citation of reference 5 suggests that it presents data about the importance of UGT in the pharmacogenomics of antipsychotics. However, UGT is only mentioned as a key word (and only as UGT-2B.. but not as UGT-1A..). In ref 6, UGT is not even mentioned. Therefore, the reader with limited knowledge in this field may not get a full picture of the importance of the subject. In summary, the Introduction should be extended!\" We sincerely apologize for inaccurate citations. In accordance with this comment and the comment from the other reviewer, introduction section is now expended to shortly summarize all gene candidates potentially important for the pharmacogenomics of antipsychotic drug pharmacokinetics. \"Table 1 presents in “Intervention I” the different phenotypes using the example risperidone. The reader is then supposed to presume that this presentation is also valid for UGT enzymes (eg gene-drug pair: UGT-olanzapine), but this classification (poor, intermediate, extensive, ultrarapid) is seemingly not introduced for these polymorphisms. If however this is the case, this should be presented in some details and corresponding references should be presented.\" We agree, Table 1 can be misinterpreted. In the introduction section, it is now described which genes have established metabolizer status definitions and which genes are instead analyzed on the basis of genotypes and haplotypes. Also, Table 1 is now updated to include another example that involves UGT isoenzyme to further eliminate confusion. \"Indeed, the average reader will not be familiar with the genetic polymorphisms of conjugating enzymes, and in particular, with their relevance for antipsychotics. In other words: are there PM, EM, UM, IM in patients treated with e.g. asenapine or olanzapine, with regard to the metabolism by UGT-enzymes? (Admittedly, in the suppl. material is some information, but the protocol should be more precise, especially in Introduction). It is a fact that among cytochrome P-450 isozymes, only about half a dozen isozymes play a role in the metabolism of antipsychotics. In contrast, there are considerably more isozymes which contribute to the conjugation of drugs, but their individual role in the metabolism of antipsychotics is poorly characterized.\" In line with this and previous comments, introduction section is now extended to also include this information. Also, our justification for the choice of UGT isoenzymes included in the first iteration of our living systematic review is included in the section 2.2. If the Reviewer finds that some crucial UGT or CYP450 isoenzymes are omitted, we will gladly consider them for the inclusion. \"The pioneering study of Haslemo et al. (CPT, 2012, 221) (besides that of Erickson et al., Pharmacogen Genom 2011, 539) shows that plasma concentrations of conjugated olanzapine (10-N-ola-glucuronide) differ in olanzapine treated patients classified according to their UGT1A genotypes, but the difference is negligible for (unconjugated) olanzapine concentrations. This rises the question whether in the present meta-analysis study both ola and conjugated ola concentrations will be considered. It could be then of interest to examine the relationships between the UGT genotypes and the ratios parent compound/metabolite (Ola/10-N-ola-glucoronide).This approach may also be valuable for all other antipsychotics, whether they are metabolized by cytochrome P-450 or by polymorphic conjugating enzymes, or by both types (e.g. risp/9-OH-risp; 9-OH-risp/conjug. 9-OH-risp; etc).\" To our knowledge, Olanzapine metabolites including 10-N-Olanzapine-Glucuronide have marginal activity and it is not recommended to measure them during therapeutic drug monitoring [PMID: 1683407]. Even though the pharmacokinetics of inactive drug metabolites is very relevant and important topic, we find this data to be of limited usefulness for our living systematic review as we plan to focus on the clinical aspects of therapeutic drug monitoring. Regarding risperidone, there is evidence that risperidone and 9-OH-risperidone have different efficacy and safety profiles due to different blood-brain-barriers permeability [PMID: 33054667]. Therefore, we agree for that analysis of riperidone/9-OH-risperidone ratio may be very useful for the interpretation of meta-analysis of risperidone active moiety (Risperidone + 9-OH-Risperidone) plasma levels. Section 2.4 is now updated to reflect this. \"Probably of little importance for the establishment of the protocol, it is to mention that in the case of risperidone, its hydroxylation to 9-OH-risperidone is stereoselective, in that CYP2D6 preferentially forms (+)-9-OH-risperidone rather than (-)-9-OH-risperidone (Yasui-Furukori et al. Drug Metab. Disp 2001, 1263). It should be considered, as rarely, stereoselective procedures were used in the published studies, the net effect of the genotype may be masked when non-stereoselective procedures are used.\" As we mentioned previously, we would like to focus on clinical aspects of therapeutic drug monitoring, i.e. we will try to use drug blood levels as a biomarkers of drugs biological effects. Therefore, even though stereo-selective analysis highlights the influence of CYP2D6, we find very limited clinical relevance of this information for our review as two enantiomers of 9-OH-Risperidone have been previously described as equipotent (PMID: 17344104). \"The authors exclude some antipsychotics, but the mentioned reasons are not always evident, and again, the citations do not always correspond to the statements. Therefore, the presentation of the reference list demands to be improved. Eg, 2.2. Drug gene pairs …: “Lurasidone will not be analyzed because of the uncertain importance of drug-blood level measurements.” Ref 6 is cited, but this ref does apparently not deal with this subject. Actually, lurasidone is mainly a substrate of CYP3A4/5. As its blood levels strongly depend on dietary conditions, this may be an important reason for recommending TDM (cf Preskorn 2013: Lurasidone should be administered with food-at least 350 kcal-to ensure maximum exposure). However, one can agree with the decision of the authors, as this drug belongs to the group of drugs mentioned in 2.5. VII (study inclusion citeria) resp. 2.6. V.\" We agree with the reviewer, Lurasidone is the substrate for CYP3A4/5 and should therefore be included. Also, unrelated to reviewer’s comment, we have noticed that we listed Paliperidone as CYP3A4/5 and CYP2D6 substrate even though paliperidone is minimally metabolized in the liver and is mostly eliminated by kidneys [PMID: 20118446]. We sincerely apologize for these errors; they are now corrected in the revised version of the manuscript. \"2.4. Definition of the outcome As pointed out by the authors, weight corrected drug concentrations may be advantageous, but they are often not available. Steady-state plasma concentrations: In the case of risperidone, there may be a problem in that in extensive metabolizers (CYP2D6), its T1/2 = 2 - 4h, but in poor metabolizers, considerably longer, comparable to that of its metabolite 9-OH-risperidone. This means that in EM, risp. steady-state concentrations will hardly be reached in EM but only in PM.\" We agree, people with lower enzyme metabolism capacities might take longer time to reach steady state. Therefore, we changed the criterion for the time needed to reach steady state to be 5x the conservative t1/2 estimate. In other words, whenever the t1/2 is presented as the range in AGNP guidelines (PMID: 28910830) we will use the higher limit of this range (longest time). This is now reflected in section 2.4. \"Extended data: March : 6. UGT1A4 “At the time of writing, there is no consensus regarding the classification of different UGT1A4 diplotypes into metabolizer groups. Therefore, pragmatically, alleles known to affect CYP1A2 activity with Minor allelic frequencies >1% in the world population will be considered for this analysis.” Not clear: this suggests that there is a relationship CYP1A2 – UGT1A4 (?).\" This was a typing error, and we sincerely apologize for it. We also noticed several other typing errors in the Extended data and they are now corrected in the new version. We again thank the Reviewer for the constructive criticism. We have no further comments." } ] } ]	1	https://f1000research.com/articles/13-452
https://f1000research.com/articles/12-344/v1	28 Mar 23	{ "type": "Case Report", "title": "Case Report: Postsurgical hallux varus in which metatarsophalangeal joint arthrodesis was useful", "authors": [ "Naoki Kondo", "Tetsuya Igarashi", "Tomoya Inukai", "Hiroyuki Kawashima", "Tetsuya Igarashi", "Tomoya Inukai", "Hiroyuki Kawashima" ], "abstract": "A 74-year-old Japanese woman who underwent Mann’s procedure with fibular sesamoidectomy for left hallux valgus 21 years ago complained of left hallucis pain. She was diagnosed with iatrogenic hallux varus and hammer toe deformities. Metatarsophalangeal joint arthrodesis and shortening oblique osteotomy were performed. After surgery, the hallux valgus angle improved from -28° to 0°, and the intermetatarsal angle between the first and the second metatarsus improved from 0° to 6°. The Japanese Society for Surgery of the Foot score improved from 75 to 81 points. She could walk without pain and sustained no deformity at 4 years after the surgery.", "keywords": [ "Hallux valgus", "Metatarsophalangeal joint arthrodesis", "Iatrogenic hallux varus", "Cup and cone reamer", "Shortening oblique osteotomy" ], "content": "Introduction\n\nHallux varus is a rare foot deformity due to iatrogenic, post-traumatic, idiopathic, inflammatory, spontaneous, or congenital pathologies. In particular, the iatrogenic type is the most common cause of hallux varus.1,2 Multiple studies reported that postsurgical hallux varus was observed in 2%–15.4% of cases.3\n\nPost-surgically observed hallux varus is attributed to overcorrection of the hallux valgus deformity. This includes excessive removal of the medial osteophyte and over-release of adductor halluces tendons, transmetatarsal ligament, and lateral metatarsophalangeal (MTP) joint capsule.1 The incidence of iatrogenic hallux varus after surgery of hallux valgus is very low, and there is a paucity of reports associated with the treatment strategy.\n\nHerein, we report a novel case of postsurgical hallux varus deformity. We performed revision surgery, i.e., MTP joint arthrodesis for hallucis and shortening oblique osteotomy for the lesser toes. Four years after surgery, the patient was satisfied, functionally good, and experienced no pain upon standing or walking. No postoperative callosity was detected.\n\n\nCase report\n\nA 74-year-old Japanese woman visited our clinic with complaints of left hallucis pain and concerns about medial deviation. Twenty-one years prior, the patient underwent Mann’s procedure with bunionectomy and with fibular sesamoidectomy, a surgical operation for bilateral hallux valgus. After the operation, the MTP joint surface deviated medially in a hyperextended position (Figure 1A–C). In addition, the second and fourth toes demonstrated hammer toe deformities (Figure 1A and C). First, an orthosis was applied; however, medial deviation and pain in her MTP joint worsened six months after the orthosis application. Hence, surgical intervention was decided on. Upon physical examination, the left MTP joint of the patient was swollen, tender, and erythematous. Extensor hallucis longus was very tense, the MTP joint was hyperextended, and the interphalangeal (IP) joint was in a flexed position, resulting in the “cock-up deformity” of hallux varus (Figure 2A–D). Lateral instability of the MTP joint of the hallux was not detected.\n\nThe extensor hallux longus is very tense, with the metatarsophalangeal (MTP) joint hyperextended and the interphalangeal (IP) joint in a flexed position, forming a so-called “cock-up deformity” (A). In the lateral view, a preoperative operation scar is clearly detected (B). In the plantar view, the varus deformity is clearly observed, and no callosity is detected (C).\n\nThe passive extension and flexion of the MTP joint are 90° (A) and -10° (B), respectively.\n\nThe passive extension and flexion of the IP joint are -20° (C) and 90° (D), respectively.\n\nThe Japanese Society for Surgery of the Foot (JSSF) hallucis score4 is often used in Japan as a means to evaluate the function of the ankle and foot. The patient’s score was 75 out of 100 points (pain, 30 out of 30; deformity, 17 out of 25; range of motion, 15 out of 15; gait, 10 out of 20; and activity of daily life, 3 out of 10).\n\nRegarding the range of motion, the MTP joint was very stiff and showed extension contracture (-90° in extension and -30° in flexion), but the IP joint remained flexible (-5° in extension and 90° in flexion) (Figure 2A–D).\n\nRadiographs showed that the hallux valgus angle (HVA) was -28° (Figure 3A). The intermetatarsal angle between the first and the second metatarsus (M1M2A) was 0° (normal range, 6°–9°), which meant that the first and second metatarsal bones were parallel (Figure 3A). As the tibial sesamoid shifted medially, and the fibular sesamoid was absent, excessive medial eminence resection might have been performed. An oblique view of the foot demonstrated that the proximal phalanx subluxated dorsally (Figure 3B).\n\nRadiographs show that the hallux valgus angle was -28°.\n\nThe intermetatarsal angle, which should be approximately 6°–9°, is 0°. This indicates that the first and second metatarsal bones are parallel.\n\nThe tibial sesamoid has shifted medially and the fibular sesamoid is absent. Excessive medial eminence resection might have been performed.\n\nIn this case, MTP joint arthrodesis, medial capsular release, and EHL tendon lengthening were performed. For the second and fifth toes, a shortening oblique osteotomy was performed. The intraoperative macroscopic findings revealed that the medial portion or the articular surface was impacted by the severe degenerative change. The degenerative changes were also observed in the capsule (Figure 4).\n\nThe medial portion of the articular surface reveals severe degenerative change.\n\nDegenerative change is also seen in the capsule.\n\nA cup and cone type reamer (Wright Medical, Tokyo, Japan) was used to preserve the length of the hallux as much as possible. The metatarsal articular surface was reamed to a cup-shaped surface, and the proximal phalanx articular surface was recreated with a cone-shape.\n\nThe hallux valgus angle was fixed at 0°, and the first proximal phalanx axis was dorsally fixed at 15° to the metatarsal bone axis. Two full-thread Acutrak® screws (Nihon Medical Next Co. Ltd, Tokyo, Japan) were inserted at the fixed position in a crisscross fashion. For the lesser toes, a shortening oblique osteotomy was performed (Figure 5A and B). The postoperative radiographs showed that M1M2A was 6° and HVA was 0° (Figure 5A).\n\nA cup and cone reamer are used to preserve the length of the hallux as much as possible. The hallux valgus angle is fixed at 0°, and the 1st proximal phalanx is dorsally fixed at 15° to the metatarsal bone. M1M2A shows 6°. For the lesser toes, a shortening oblique osteotomy is performed (A, anteroposterior view; B, oblique view).\n\nTemporal fixation of the lesser toes with Kirchner wires (1.2 mm in diameter) was performed, and then the wires were removed after three weeks. Arch support was applied, and full weight gait exercise was performed.\n\nThe screws remained intact and in place, and no valgus or varus deformities were apparent four years after surgery (Figure 6A–C).\n\nNo recurrence of hallux varus and hallux valgus is observed (A). The screws are intact, in place, and no valgus or varus deformities are apparent. Additionally, each osteotomized lesser toe is united (B, anteroposterior view; C, oblique view).\n\nPost-operative evaluation of the JSSF score of hallucis was 81 out of 100 (pain, 30 out of 30; deformity, 23 out of 30; range of motion, 5 out of 15; gait, 20 out of 20; and activity of daily life, 3 out of 10), which showed a six-point increase. A six-point increase in deformity and a 10-point increase in walking abilities were noted; however, a 10-point decrease in range of motion was observed. No deformities were apparent and pain that worsened during movement was relieved.\n\n\nDiscussion\n\nHallux varus is a clinical condition characterized by the medial deviation of the great toe at the MTP joint. Iatrogenic hallux varus is caused by an imbalance between the various bone, tendon, and capsule-ligament structures of the first MTP joint, including a progressive medial deviation of the hallux.5\n\nThe causes of iatrogenic hallux varus are 1) overstitching of the medial joint capsule, 2) medial deviation of the tibial sesamoid, 3) over-traction by the abductor hallucis muscle due to lateral ligament complex release, 4) postoperative dressing in varus position of the hallux metatarsophalangeal joint, and 5) over-excision of the medial bony protrusion of hallux metatarsus. The patient in our case exhibited the second and fifth causes.6\n\nAkhtah et al. reported that the hallux varus was mainly classified into three types: osseous, myoligamentous, and combined.7 In our case, the hallux varus was the combined type.\n\nThe surgical procedures for iatrogenic hallux varus depend on salvaging the MTP joint of the hallucis. When the MTP joint can be preserved, the medial joint capsule is released, and the MTP joint is reconstructed using procedures such as reverse chevron osteotomy and tendon transfer of abductor hallucis.6\n\nLeemrijse et al. recently reported a treatment algorithm.5 According to this algorithm, in the case of a stiff MTP and mobile IP joint of the hallux, MTP joint arthrodesis is indicated. The present case also demonstrated both stiff MTP and mobile IP. Subsequently, MTP joint arthrodesis was performed.\n\nWhen MTP cannot be preserved, MTP joint arthrodesis is indicated; however, MTP joint mobility is sacrificed. In this case, severe joint incongruity and irreversible flexion contracture of the MTP joint were detected. In addition, intraoperative findings revealed that the MTP joint surface degenerated at both the metatarsal and proximal phalanx articular surfaces (Figure 4). Therefore, MTP joint arthrodesis was selected.\n\nLeemrijse et al. and Piat et al. described that MTP fusion is the most reliable solution and is inevitable if the joint is stiff or degenerative.5,8\n\nMTP joint arthrodesis for hallux varus also significantly improved both the average 1–2 intermetatarsal angle from 4.8° to 8.4° and HVA from -20.7° to 8.1° in 26 patients (29 feet).9 In our case, M1M2 A improved from 0° to 6° and HVA improved from -28° to 0° postoperatively (Figures 3A and 5A).\n\nTourne et al. reported 14 cases of hallux varus. Each case showed medial arthrolysis of the MTP joint. Of 14 patients, five were treated with a reconstruction procedure of the lateral ligament accompanied by the medial release. Thereafter, nine patients were treated with MTP joint arthrodesis in case the MTP joint was stiff and arthrosis was present. According to the 100-point scoring system, the results were excellent in 56% and good in 44% of the patients with MTP joint arthrodesis.10\n\nThe guidelines for our cases were as follows: 1) V-shaped incision was used for the dorsal MTP joint capsule. Thereafter, the articular surface was sufficiently exposed and medial tightness was thoroughly released. Fibular sesamoid was also released and relocated; subsequently, the V-shaped flap was tightly repaired after the MTP joint fusion. 2) The MTP joint level of hallucis after the primary surgery was much shorter than those of the lesser toes. To correct the imbalance of the MTP joint line between hallucis and lesser toes, and to prevent postsurgical metatarsalgia, we used a cup and cone reamer to minimize bone excision of the hallux metatarsus. Ball and cup reamer and osteosynthesis with pure titanium staples have been reported to yield good results in 54 patients with hallux valgus.11 In addition, we performed shortening oblique osteotomy for lesser toes. 3) EHL elongation was performed because the EHL tendon became shortened due to flexion contracture of hallucis.\n\nIn conclusion, MTP joint arthrodesis using a cup and cone reamer minimized the shortening length of the metatarsal bone and proximal phalanx bone of the hallux. Additionally, it enabled stabilization in walking and bearing on the foot, resulting in good functional outcomes for this iatrogenic hallux varus case.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe authors acknowledge Professor Yasuhito Tanaka (Division of Orthopedic Surgery, Nara Medical University) and Professor Go Omori (Niigata University of Health and Welfare) for their technical supervision and instructions.\n\n\nReferences\n\nMohan R, Dhotare SV, Morgan SS: Hallux varus: A literature review. Foot (Edinb.). 2021; 49: 101863. PubMed Abstract \| Publisher Full Text\n\nDonley BG: Acquired hallux varus. Foot Ankle Int. 1997; 18: 586–592. Publisher Full Text\n\nHawkins FB: Acquired hallux varus: cause, prevention, and correction. Clin. Orthop. Relat. Res. 1971; 76: 169–176. Publisher Full Text\n\nNiki H, Aoki H, Inokuchi S, et al.: Development and reliability of a standard rating system for outcome measurement of foot and ankle disorders I: development of standard rating system. J. Orthop. Sci. 2005; 10: 457–465. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeemrijse T, Bevernage BD: Surgical treatment of iatrogenic hallux varus. Orthop. Traumatol. Surg. Res. 2020; 106: S159–S170. Publisher Full Text\n\nCoughlin MJ, Salzman CL, Anderson RB: Mann’s Surgery of the Foot and Ankle. 9th ed.Philadelphia, PA: Elsevier/Saunders; 2014; 301–309.\n\nAkhtah S, Malek S, Hariharan K: Hallux varus following scarf osteotomy. Foot (Edinb.). 2016; 29: 1–5. Publisher Full Text\n\nPiat C, Cazeau C, Stiglitz Y: Post-operative hallux varus: a review of treatment methods. Int. Orthop. 2021; 45: 2193–2199. PubMed Abstract \| Publisher Full Text\n\nGeaney LE, Myerson MS: Radiographic results after hallux metatarsophalangeal joint arthrodesis for hallux varus. Foot Ankle Int. 2015; 36: 391–394. PubMed Abstract \| Publisher Full Text\n\nTourne Y, Saragalia D, Picard F, et al.: Iatrogenic hallux varus surgical procedure: a study for 14 cases. Foot Ankle Int. 1995; 16: 457–463. PubMed Abstract \| Publisher Full Text\n\nBesse JL, Chouteau J, Laptoiu D: Arthrodesis of the first metatarsophalangeal joint with ball and cup reamers and osteosynthesis with pure titanium staples Radiological evaluation of a continuous series of 54 cases. Foot Ankle Surg. 2010; 16: 32–37. PubMed Abstract \| Publisher Full Text" }	[ { "id": "236596", "date": "08 Feb 2024", "name": "Jun-Ichi Fukushi", "expertise": [ "Reviewer Expertise Foot and ankle 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\nThe report describes a case with postsurgical hallux varus, which can be a challenging issue for foot and ankle surgeons. This case report is overall well written, and the reviewer understands the usefulness of MTP joint arthrodesis as a reliable solution for iatrogenic hallux varus.\n\nPoints of reviews.\nIntroduction 1.In the first paragraph, the authors documented the incidence of postsurgical hallux varus as 2%-15.4%, which does not seem like “very low” to the reviewer. Therefore, the authors are required to reconsider the expression regarding the incidence of iatrogenic hallux varus in the second paragraph.\nCase report 1.JSSF hallux scale consists of three major items, which are pain, function, and alignment. The JSSF scale used in the present study consists of five items, including pain, deformity, ROM, gait, and ADL, which are used for JSSF RA foot and ankle scale (Ref 1). The authors are required to explain the reason why they used JSSF RA scale instead of hallux scale.\n2.How did the authors evaluate ROM of the hallux? Although the authors mentioned as “the MTP joint was very stiff” and “extension contracture”, the item for ROM was 15 (out of 15 points) preoperatively. The flexion of the hallux was mentioned as -10 in the legend of figure 2, and -30 in the main text.\n3.In the 7th paragraph, it is also desirable to address the documentation regarding lesser toe shortening osteotomy in more detail.\nDiscussion 1.The third paragraph (starting with “Akhtah et al”..) consisting of two sentences may be unnecessary.\n2.In the following four paragraphs (4th to 7th), salvage procedures for iatrogenic hallux varus were discussed. However, some of the description referring Leemrijse et al was redundant. Adding the documentation of the indication for MTP preservation as well as MTP arthrodesis will provide readers with a better understanding of treatment algorithm advocated by Leemrijse.\n3.Was there fibular sesamoid remained? In the paragraph 10, the authors mentioned as “Fibular sesamoid was also released..” On the other hand, fibular sesamoidectomy was supposed to be performed 21 years prior to the revision surgery.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "11458", "date": "09 Jul 2024", "name": "Naoki Kondo", "role": "Author Response", "response": "Points of reviews. Introduction 1.In the first paragraph, the authors documented the incidence of postsurgical hallux varus as 2%-15.4%, which does not seem like “very low” to the reviewer. Therefore, the authors are required to reconsider the expression regarding the incidence of iatrogenic hallux varus in the second paragraph. 　 →（Response）　I corrected from “very low” to “ not very low” in the revised manuscript. Case report 1.JSSF hallux scale consists of three major items, which are pain, function, and alignment. The JSSF scale used in the present study consists of five items, including pain, deformity, ROM, gait, and ADL, which are used for JSSF RA foot and ankle scale (Ref 1). The authors are required to explain the reason why they used JSSF RA scale instead of hallux scale. →（Response）　In the present case, we evaluated JSSF RA foot and ankle scale for the functional outcome because the surgery was performed in not only hallux but also lesser toes. 2.How did the authors evaluate ROM of the hallux? Although the authors mentioned as “the MTP joint was very stiff” and “extension contracture”, the item for ROM was 15 (out of 15 points) preoperatively. The flexion of the hallux was mentioned as -10 in the legend of figure 2, and -30 in the main text. →（Response） Thank you so much for the precious comment. We reconfirmed the manuscript and legends related to range of motion of MTP joint (preoperatively). Actually, passive extension of MTP joint was 90 °and passive flexion was -30 °. The range of motion was 60°, so this condition was neither “stiff” nor “extension contracture” but “limited range of motion”. So, we corrected the legend of Figure 2 from “-10°“ to “-30°”. In addition, the item of ROM was corrected from 15 (out of 15 points) to 13. The range of motion of IP joint was also corrected in the manuscript as follows; “,but the IP joint remained flexible (-20°in extension and 90°in flexion)(Figures 2A-2D). 3.In the 7th paragraph, it is also desirable to address the documentation regarding lesser toe shortening osteotomy in more detail. →（Response）　Dorsal curved skin incisions were applied in between the 2nd and the 3rd metatarsals and between the 4th and 5th metatarsals. Wounds were deepened and extensor digitorum brevis tendons were identified and partially resected. Distal metatarsal regions were subperiosteally released and shortening oblique osteotomy were performed with 45-degrees tilted to the metatarsal longitudinal axis for the 2nd through 5th toes. The osteotomized thickness were 7 mm. Then, distal fragment was flipped dorsally and osteophyte in the metatarsal head was totally removed and rasped. A Kirschner wire of 1.2 mm in diameter was inserted into the metatarsal, and proximal phalanx, mid phalanx, and distal phalanx for each toe. Three weeks after the insertion, these wires were removed and weight bearing and gait exercises were performed using arch support. Discussion 1.The third paragraph (starting with “Akhtah et al”..) consisting of two sentences may be unnecessary. → We deleted the 3rd paragraph in Discussion part. 2.　In the following four paragraphs (4th to 7th), salvage procedures for iatrogenic hallux varus were discussed. However, some of the description referring Leemrijse et al was redundant. Adding the documentation of the indication for MTP preservation as well as MTP arthrodesis will provide readers with a better understanding of treatment algorithm advocated by Leemrijse. → （Response）Thank you so much for the precious comment. I corrected the part as follows; “For procedures of MTP joint preservation, soft tissue release and tendon transfer method using extensor hallucis longus tendon or abductor hallucis longus tendon are used. For soft tissue release, medial capsule release or intermetatarsal space release For tendon transfers, 2 methods such as a dynamic transfer and a static transfer are known. The dynamic transfer means transfer with muscle body and the static transfer means transfer with out muscle body. Both transfers compensate for the incompetent lateral collateral ligament. Extensor hallucis longus tendon transfer is selected for a dynamic transfer with or without interphalangeal joint fusion. Abductor hallucis tendon transfer is also used. For static tendon transfer, abductor hallucis tendon or artificial implant (TightRope) is selected.　 When MTP joint is unstable or limited range of motion, MTP joint cannot be preserved , so MTP joint arthrodesis is selected. The present case demonstrated that MTP joint congruity was not so good because of the joint instability and that MTP joint degeneration was clearly detected (Figure 4). Therefore, MTP joint arthrodesis was performed. “. 3.Was there fibular sesamoid remained? In the paragraph 10, the authors mentioned as “Fibular sesamoid was also released..” On the other hand, fibular sesamoidectomy was supposed to be performed 21 years prior to the revision surgery. →　(Response) Thank you so much for the comment. I reconfirmed the operative record. I corrected from fibular sesamoid to tibial sesamoid. The released sesamoid was tibial one." } ] }, { "id": "192634", "date": "14 Jun 2024", "name": "Inderjeet Singh Rikhraj", "expertise": [ "Reviewer Expertise Outcomes 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\nWell written report on a rare complication. Highlights the problem of an iatrogenic hallux varus due to resection of the fibula sesamoid. The JSSF score have improved. It would be good to highlight the Minimal Clinical Important Difference (MCID) of the JSSF to point out the significance of the improvement.\nIn the abstract, it is stated that the fibular sesamoidectomy was done 21 years ago. However, in the discussion, paragraph 10, line 2, it is stated that the fibular sesamoid was released and relocated. How is it possible to do this when the fibular sesamoid was resected?\nIntroduction A medial osteophyte is not removed, it is a bunionectomy.\nCase Report \"First, an orthosis was applied; however, medial deviation and pain in her MTP joint worsened six months after the orthosis application\" Please rewrite and put a full top and not a colon. \"As the tibial sesamoid shifted medially, and the fibular sesamoid was absent, excessive medial eminence resection might have been performed\" This could also result from erosion as the proximal phalanx is abutting the medial aspect of the first metatarsal head, as the index surgery was done many years ago\nDiscussion \"The surgical procedures for iatrogenic hallux varus depend on salvaging the MTP joint of the hallucis.\" this sentence is redundant. Para 2 \" The patient in our case exhibited the second and fifth causes\" what does this line mean? Combine paras 4 & 5 and rephrase as there is repetition and rewrite it Para 10 should be removed and put under Case Report. It is only 1 case report, it cannot be plural. In Abstract and Introduction, you state that a fibular sesamoidectomy was performed in the initial surgery. How can your surgical procedure state \"Fibular sesamoid was also released and relocated\" Last para \"Additionally, it enabled stabilization in walking and bearing on the foot\" There should be the words \"weight -bearing\" and not \" bearing\" This sentence should be rewritten to state that the foot was stable in weightbearing and walking\nThe surgery was done well especially of the lesser toes.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "11790", "date": "09 Jul 2024", "name": "Naoki Kondo", "role": "Author Response", "response": "I appreciate your precious comment.　 I reconfirmed the operative record. As you pointed out, fibular sesamoidectomy had been performed. I corrected from fibular sesamoid to tibial sesamoid In Discussion part. The released sesamoid was tibial one. Including the reviewer comment 1, I totally revised the manuscript and I showed the corrected parts were colored with red." } ] } ]	1	https://f1000research.com/articles/12-344
https://f1000research.com/articles/13-775/v1	08 Jul 24	{ "type": "Study Protocol", "title": "Biofeedback therapy using Cerebri for the prevention of migraine attacks in adults with episodic migraine (BioCer): a randomized, wait-list controlled trial – the study protocol", "authors": [ "Amalie Christine Poole", "Anker Stubberud", "Melanie Simpson", "Lise Rystad Øie", "Einar Tobias Vassbø Skalstad", "Marte-Helene Bjørk", "Espen Saxhaug Kristoffersen", "Kjersti Grøtta Vetvik", "Alexander Olsen", "Iben Cornelia Keim Larsen", "Mattias Linde", "Erling Andreas Tronvik", "Tore Wergeland Meisingset", "Anker Stubberud", "Melanie Simpson", "Lise Rystad Øie", "Einar Tobias Vassbø Skalstad", "Marte-Helene Bjørk", "Espen Saxhaug Kristoffersen", "Kjersti Grøtta Vetvik", "Alexander Olsen", "Iben Cornelia Keim Larsen", "Mattias Linde", "Erling Andreas Tronvik", "Tore Wergeland Meisingset" ], "abstract": "Introduction Biofeedback is a non-pharmacological treatment option valued for its minimal risk of adverse events and offers a safe alternative for individuals seeking preventive care for migraine. Despite level A evidence for migraine prevention, biofeedback treatment is still unavailable to most patients. We developed a novel medical device (Cerebri) for multimodal biofeedback treatment that omits the need for healthcare personnel involvement. Cerebri consists of a smartphone application (app) and two wireless sensors. Unique in its approach, the Cerebri app seamlessly integrates three biofeedback modalities – heart rate variability, temperature, and electromyography – making it a comprehensive, therapist-independent solution for non-pharmacological migraine management.\n\nMethods Biofeedback therapy using Cerebri for the prevention of migraine attacks in adults with episodic migraine (The BioCer study) was an open-label, randomized, waitlist-controlled, multicenter trial. This study investigated the safety and efficacy of daily home-based biofeedback sessions using the Cerebri device. A total of 286 participants will be randomized to either a 12-week intervention arm or waitlist control arm. The primary outcome was the change in the mean number of migraine days from baseline to the last 28-day period during the treatment phase in the treatment group compared with the control group. The primary outcome variable was prospectively collected through daily eDiary entries.\n\nEthics and Dissemination Approval from the ethics committee and competent authorities was obtained prior to study initiation. Participation was voluntary and informed and written consent was obtained prior to inclusion. The results of this trial will be published in peer-reviewed international medical journals and communicated to patients and healthcare personnel through the relevant channels.\n\nTrial registration numbers EUDAMED: CIV-NO-22-08-040446 REK (Regional Committees for Medical and Health Research Ethics): 502734 Date of approval 2022-10-14 Trial registration: NCT05616741, 2022-11-15, https://clinicaltrials.gov/study/NCT05616741", "keywords": [ "mHealth", "biofeedback", "behavioural therapy", "headache", "migraine", "neurology" ], "content": "Introduction\n\nMigraine is the second most disabling disorder worldwide and incurs enormous societal costs, estimated to exceed €50 billion per year in the EU alone.1,2 Frequent migraine attacks warrant preventive treatment. A multitude of preventive pharmacological options are available; however, there are several barriers to effective treatment. For instance, one study reported that over two-thirds of patients discontinued their prophylactic migraine medication after six months, and more than 80% after a year, due to either a lack of efficacy or adverse reactions.3\n\nSeveral non-pharmacological behavioral interventions for migraine, such as biofeedback, have been highlighted as non-invasive prophylactic treatment regimens with a low risk of negative side effects, which can be an alternative or adjunct to pharmacological therapies. Biofeedback combined with relaxation training is listed with Grade A level evidence for migraine prevention in the 2000 guidelines from the American Academy of Neurology.4 This has been continued in the updated 2021 American Headache Society consensus statement, which also suggests mobile health (mHealth) application-based approaches for delivering biobehavioural treatments to increase patient access and participation.5\n\nBiofeedback utilizes technologies and equipment to monitor physiological processes that are usually considered involuntary and modulated without conscious awareness. Presented with real-time feedback on these processes, users can learn to exercise control over them, which in turn has a beneficial effect on the migraine symptom burden. Despite its effectiveness, biofeedback has limited accessibility. The high financial costs of having a therapist instructing the procedure, necessary equipment, and patient travelling costs are likely to contribute to this. To our knowledge, no therapist-independent migraine biofeedback treatment with proven efficacy is currently available.6\n\nTo address this gap, a multimodal biofeedback system, the Cerebri system, has been developed for therapist-independent home-based use. Unique to this product, the Cerebri application (app) seamlessly integrates three biofeedback modalities: heart rate variability (HRV), temperature, and electromyography (EMG), making it a comprehensive, home-based solution for non-pharmacological migraine management. The device is a product of a thorough development process, including a hardware feasibility study7 and three development studies on progenitor versions of the system with short test periods.8–10 In addition, prior to the initiation of this trial, a feasibility study with 3 month follow-up was performed.11\n\n\nProtocol\n\nThe objective of this study was to investigate the efficacy and safety of a 12-week daily use of the Cerebri biofeedback device as compared to a waitlist control in adults with episodic migraine.\n\nPrimary efficacy endpoint\n\nThe primary endpoint was the change in the mean number of migraine days from baseline to the last 28-day period (week 9-12) during the treatment phase in the treatment group compared to the wait-list control group.\n\nSecondary efficacy endpoints\n\nThe key secondary endpoint analyses were changes in mean number of migraine days in week 1-4 and week 5-8, change in mean migraine intensity, proportion of 30% responders, change in acute migraine drug treatment use, and change in subject-reported headache-related disability. Exploratory and descriptive endpoints included changes in weekly migraine days, mean headache days per 28-day period, eDiary and biofeedback session adherence, and mean Patient Global Impression of Change (PGI-C) scores. Additionally, changes in the mean number of migraine days during the extended biofeedback period were assessed.\n\nSafety endpoints\n\nDescription of the frequency and severity of treatment-emergent adverse events (AEs), adverse device effects (ADEs), serious adverse device effects (SADEs), and unexpected serious adverse side effects (USADEs). In the case of pregnant participants, at inclusion or acknowledged during participation, no additional monitoring of maternal or fetal health was instituted. In such cases, participants will be asked if they agree to be followed up until the end of pregnancy to collect data on any pregnancy complications and fetal outcome data.\n\n\nMethods and analysis\n\nThis was an open-label, randomized, waitlist-controlled, multicenter trial. The study design followed the standard for clinical investigation of medical devices for human subjects – good clinical practice (ISO 14155:2020) – and the relevant trial guidelines of the International Headache Society.12 Reporting follows the CONSORT guidelines.\n\nThe total duration of study participation was 16 weeks, with the possibility of a 12-week extension period (Figure 1). After the screening visit, if eligible, the participants were assigned to a baseline period of a minimum of four weeks in accordance with the guidelines.12 During the baseline period, participants were asked to maintain a daily headache diary in the Cerebri smartphone app. Upon completion of the baseline period, the participants were rescreened for eligibility prior to randomization. This design, in wherein the baseline period also serves as a screening period, enables rescreening based on eDiary entries. This is the recommended approach for enrolling RCTs on episodic migraine.12 Participants deemed ineligible at the point of rescreening, who withdraw from or are lost to follow-up during the baseline period, are categorized as screening failures and will be excluded from further participation.\n\nEligible participants will be randomly allocated to the treatment or waitlist groups. The treatment group will receive the medical device by postal service, and a treatment period of 12 weeks will be initiated on the first day of completing a biofeedback session. Participants in the waitlist group completed daily eDiary entries for 12 weeks from the day of randomization.\n\nBoth arms were invited to participate for a 12-week extension period. Participants randomly allocated to the waiting list were offered the possibility of entering a 12-week extension phase with biofeedback treatment. The 12-week extension period was initiated on the first day of the biofeedback training. The participants randomized to the intervention group were invited to a 12-week extension period after completing their 12-week biofeedback treatment, in which they continued with daily eDairy only.\n\nParticipants will not have access to the intervention at the end of the study. Beyond the voluntary 12-week extension period, there will be no long-term follow-up on safety and survival status, except in cases of pregnancy.\n\nRandom group allocation will occur a minimum of four weeks after inclusion and after rescreening. If eligible, permuted block randomization was performed. Randomization was stratified on the adjunct use of preventive medication.\n\nBlinding/masking is not possible owing to the nature of the intervention in question and will thus not be performed.\n\nThis trial will recruit participants from all over Norway, with inclusion sites covering all health regions. The investigational sites were the neurological departments at St. Olavs Hospital, Trondheim University Hospital, Akershus University Hospital, Haukeland University Hospital, and University Hospital of North Norway. Visits 1 (baseline inclusion) and 2 (rescreening and randomization) was performed by medical doctors with headache expertise. All visits were conducted remotely via video or telephone. Information about the trial and contact information to indicate interest in participation was disseminated through relevant institution webpages and social media platforms. Interested parties may contact the study site directly via email or may be referred. Study personnel were prohibited from enrolling patients for whom they were primarily responsible for treatment and/or follow-up.\n\nA full list of the inclusion and exclusion criteria is listed in Table 1. Eligibility was assessed at visits 1 and 2 (Figure 1), and participants must comply with all inclusion criteria and none of the exclusion criteria to proceed to randomization. Participants were allowed to use one preventive migraine medication if they were stable upon study entry and remained unaltered during study participation. In the case of onabotulinum toxin A injections following the PREEMT injection paradigm, a stable treatment regimen was operationalised as at least three consecutive treatments prior to inclusion as part of their previous regular follow up prior to study entry. For other prophylactic treatments, a duration of three months or five half-lives, whichever is longer, of unaltered use is considered stable.\n\n\n\n1. 18 years of age or older.\n\n2. Episodic migraines with or without aura diagnosed by a neurologist/physician per International Classification of Headache Disorders 3rd edition (ICHD-3).\n\n3. History of at least 4 and up to 14 days of migraines per 28-day period in the 3 months prior to screening (as recalled by the subject). This frequency must be confirmed in the headache diary before randomization to treatment or wait-list control.\n\n4. At least three months of experience with smartphone and access to an iOS or Android phone at home.\n\n5. Capable of giving signed informed consent with includes compliance with the requirements and restrictions listed in the informed consent form (ICF) and in this protocol.\n\n6. Onset of migraine before age 50 years.\n\n\n\n7. More than 14 days of headache (all types) per 28-day period.\n\n8. Subjects diagnosed with trigeminal autonomic cephalalgias and neuralgias.\n\n9. Subjects with secondary headache conditions.\n\n10. Subjects with pathologies that inhibit use of the device according to the instructions for use (e.g. blindness, deafness).\n\n11. Use of non-pharmacological preventive treatment (meditation, physical therapy, psychotherapy as a headache treatment, acupuncture etc.) with the exception of stable treatment for other indications than migraine.\n\n12. Use of concurrent migraine preventive medication, with the exception of stable dose (≥3 months) monotherapy of migraine preventive medication.\n\n13. Subjects taking opioids (≥3 days per month) or barbiturates at the time of screening.\n\n14. Subject participates in another clinical investigation or has participated in CER-MIG-1.\n\n15. Alcohol overuse or illicit drug use.\n\n16. Subject who is unlikely to follow Clinical Investigation Plan or where treatment seems futile in the opinion of the investigator or have demonstrated an inability to sufficiently adhere to headache diary entries (<70%).\n\nThis study will be conducted in accordance with the principles of ISO 14155:2020 (Clinical Investigation of Medical Devices in Human Subjects), Good Clinical practice, and the Guidelines of the International Headache Society. The study was approved by the Regional Committees for Medical Research Ethics (REK, approval number 502734. Date of approval 2022-10-14) and the Norwegian Medical Product Agency (NOMA) (reference number 22/17254-5. Date of approval 2022-09-01) prior to initiation. Substantial protocol amendments will be reviewed by competent authorities before implementation. Data protection follows the GDPR. We adhere to the declaration of Helsinki.\n\nTo ensure that all subjects have similar opportunities to benefit from enrolment, participants randomized to the waitlist group were offered treatment following the conclusion of the waitlist period. Consent was collected by the investigator conducting the screening visit. Study information was sent in advance of visit 1 by email, scripted study information was provided orally, and any questions addressed during visit 1 prior to signing to ensure that consent was obtained in all cases. Consent was collected by a digital signature via the national electronic data entry application («Helsenorge»). Study personnel were prohibited from recruiting patients for whom they were directly responsible for treatment.\n\nInvestigational medicinal product\n\nThe medical device Cerebri consists of a smartphone app and a set of two wireless non-invasive sensors. Both sensors are skin-contact-dependent. One sensor is attached to the index finger and measures HRV and peripheral skin temperature. The second sensor is attached to the skin over the upper trapezius muscle fibers using adhesive electrodes and measures muscle tension as surface electromyographic voltage (Figure 2A and 2B). The sensors transmit the measurements to the user’s smartphone by Bluetooth technology and display them in real time to the user. By following instructions for suggested exercises displayed in the Cerebri smartphone app, the subject trains on influencing the measured parameters in the desired direction during each biofeedback session. Figure 3A-C illustrate how visualization of the physiological measures are displayed on their smartphone. Exercises to facilitate successful manipulation of the physiological parameters include breathing techniques, progressive muscle relaxation, and awareness training. A visual breathing pacer to support paced breathing is available in the mobile app to the user during the entire treatment session. It is desirable to increase HRV and peripheral skin temperature and lower neck muscle tension. Participants are instructed to sit comfortably in a quiet room during treatment. Throughout the intervention period, participants are required to complete a 10-minute biofeedback session at least once daily at a time of their choosing. They have the option to perform as many as six sessions each day.\n\neDiary and biofeedback\n\nData on headache is collected prospectively as a patient-reported outcome measure in an eDiary integrated into the Cerebri smartphone app. Daily entries are encouraged, but the app allows the entry of registrations from the two previous calendar days. The tool is designed such that when participants report headaches, they are prompted to answer additional questions. Data collected in the diary include the estimated duration of headache, intensity of headache using an 11-point scale (0-10), and the use (type, dose, and number of administrations) of acute medications (triptan, paracetamol, NSAIDs, opioids, other), and information on whether acute medications relieved headache (Table 2 and Figure 4A and 4B).\n\nA web-based dashboard solution enables study personnel to access a real-time overview of adherence to eDiary and biofeedback treatment. Thus, study personnel can identify and reach out to participants with low adherence. In the case of technical issues, a support service provided by the sponsor will be available. The dashboard also includes information regarding the migraine/headache status on a given day and acute medication use. This information will be accessible to study personnel in the baseline period for rescreening purposes. Post randomization, only the adherence data will be available.\n\nQuestionnaires\n\nSubjects will be asked to complete validated questionnaires at the end of each 28-day period to measure the headache-related impact on the quality of life and their impression of treatment efficacy. The questionnaires used in this trial are the Migraine-Specific Quality-of-Life Questionnaire (MSQ) v2.113 and the Patient Global Impression of Change (PGI-C).14 The MSQ is copyrighted and registered with the United States Library of Congress by Glaxo Wellcome, Inc. A written permission for use was obtained prior to use.\n\nElectronic patient record\n\nPatient records were the source of information regarding medical history, current medications in use, and previous trials of prophylactic migraine treatment acquired during visit 1. Alterations in health status, including treatment-emergent adverse events during trial participation are registered in the medical records.\n\nStudy-specific electronic case report form (eCRF)\n\nAn eCRF applying the WebCRF3 software (version 3) (https://webcrf3.medisin.ntnu.no) was developed for this study. The webCRF software was provided and maintained by the Unit for Applied Clinical Research (AKF) at the Faculty of Medicine and Health Sciences, NTNU. Data collected during study visits, telephone consultations, and safety information were entered. Data were stored in a pseudo-anonymous manner, and each study participant was identified using a unique study identification (ID) number. An open-source alternative OpenClinica (https://www.openclinica.com/get-free-community-edition-software) is able to perform an equivalent function as WebCRF3.\n\nStudy monitoring\n\nNo data-monitoring committee was appointed for this study. An independent Data Monitor will be used for review of the study in accordance with a Monitoring Plan.\n\nData management\n\nData management is guided by the data management plan. Data validation and verification of a minimum of 10% of participants will be conducted before data lock. Data lock can be performed when the last participant ends its 12 week post-randomization period, that is, while the participants are still in an extension period.\n\nResearch hypothesis\n\nThe primary endpoint is to investigate the change in the mean number of migraine days from baseline to the last 28-day period in the treatment group compared with the wait-list control group. Thus, the null hypothesis (H0) and alternative hypothesis (H1) to be tested in relation to the primary objective are as follows.\n\n• H0: The change in the mean number of migraine days from the 28-day baseline to the last 28-day period in the treatment group is equal to the change in the mean number of migraine days from baseline to the last 28-day period in the wait-list control group.\n\nvs.\n\n• H1: The change in the mean number of migraine days from the 28-day baseline to the last 28-day period in the treatment group is not equal to the change in the mean number of migraine days from baseline to the last 28-day period in the wait-list control group.\n\nSample size\n\nThe sample size calculation is based on an assumption of the effect size derived from a relevant meta-analysis of the efficacy of biofeedback in treating migraine15 and a randomized pilot study.9 Provided an effect size of d = 0.4, a significance level alpha of 0.05 (two-sided), power 0.8, and a dropout rate of 30% in the post-randomization period, the required sample size of this study was estimated to 286 participants. The anticipation of high dropout rates in this study is justified in the significant challenge of maintaining participant adherence observed in home-based mHealth studies.9,16\n\nProtocol deviations\n\nParticipants with incomplete or missing headache diary entries in more than 30% of days in the post-randomization period, participants with more than 30% missing or incomplete biofeedback sessions in the treatment arm, and participants initiating new or altering the use of their established preventive headache medications during the trial, are considered protocol deviators.\n\nStatistical analysis plan\n\nThe statistical analysis plan (SAP) will include a technical and detailed description of statistical analysis. Statistical analyses will be conducted by an independent statistician. The analysis will follow a predefined order, where the primary efficacy endpoint is analyzed first. The trial statistician will analyze the efficacy outcomes without knowledge of treatment allocation by using a dataset with study arm allocation coded as groups A and B.\n\nData analysis set\n\nFor the efficacy analysis, the full analysis set includes all randomized participants and constitutes the intention-to-treat (ITT) population. The per protocol (PP) population includes all randomized participants without protocol deviations.\n\nAnalysis dataset 1 is defined as the data from the 16-week period for all randomized participants and does not include extension period data. The 12-week post-randomization period in the biofeedback arm starts on the date of the first biofeedback session. Data lock for the evaluation of efficacy endpoints can be performed upon the completion of this dataset.\n\nThe safety data set includes all participants exposed to the biofeedback device.\n\nPrimary endpoint analysis\n\nAnalysis of the study endpoints will be performed in the ITT (all randomized subjects) and PP populations.\n\nThe primary endpoint variable is migraine days. The operational definition used for the analysis will be prespecified in the SAP.\n\nThe planned analysis strategy involves a mixed logistic regression model which can include all available daily eDiary entries. The mixed logistic regression model will be used to estimate the difference in the change from baseline in the mean number of migraine days per 28-day period between the treatment and waitlist groups. The model will include the presence or absence of migraine on each day with recorded information of the last 28 days of the baseline period and the 12 weeks post randomization as a dependent binary variable, treatment allocation, time period (baseline, week 1-4, week 5-8 and week 9-12) and an interaction term between treatment allocation and post-injections time-periods are included as categorical fixed effects, together with the stratification variable (concomitant preventive medication use). The patient ID will be included as a random effect. The site of inclusion will also be assessed as a potential random effect prior to the unblinding of the statistician to the treatment allocation.\n\nThe primary effect estimate will be the model-based estimates of the difference in change from baseline in mean monthly migraine days between the treatment and waitlist groups, presented with a 95% confidence interval.\n\nBaseline variables that are considered to be highly predictive of outcomes will be discussed and identified a priori and described in the SAP. These variables will be included in multivariate analysis. The SAP will also detail the analysis strategies for the secondary outcomes. Post hoc stratified analyses by sex (women vs. men) and assessment of sex interaction will be conducted.\n\nSafety data\n\nSafety objectives will be assessed by collecting data on treatment-emergent adverse events encountered during biofeedback training, including adverse events (AEs), serious adverse events (SAEs), adverse device effects (ADEs), serious adverse device effects (SADEs), and unanticipated SADEs (USADEs), and by evaluating the frequency and severity of these occurrences.\n\nAll data on treatment-emergent adverse events, ADEs, SADEs, and USADEs will be categorized and summarized. The frequency and severity of treatment-emergent AEs, ADEs, SADEs, and USADEs will be summarized for all enrolled participants and the entire duration of the intervention. Adverse events occurring more than once in individual participants will also be summarized and presented descriptively.\n\nThe results of this trial will be published in peer-reviewed international scientific journals. The aim is for results to be made available and spread widely to the public, general practitioners, neurologists, and other health professionals who treat patients with headache. The results will be communicated to patients through relevant channels, such as social media, patient organizations, journals for popular science, and appropriate events and conferences.\n\nStudy start 01.01.2023.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first pivotal randomized controlled trial (RCT) with an adequate sample size to properly evaluate the efficacy of home-based biofeedback treatment in patients with episodic migraine. A 2022 systematic review of digital headache interventions identified only two RCTs evaluating biofeedback apps for migraine.17 Both were pilot studies with small sample sizes and did not show statistically significant between-group differences in important headache related outcomes.9,16\n\nThe uniqueness of Cerebri is that it combines three different biofeedback modalities, whereas traditional treatments typically use one parameter.15,18 In addition, recent app-based solutions have applied one modality.16,19,20 Different modalities may benefit different users; for example, some users might find it easier to influence their finger temperature, whereas others might prefer muscle tension as their main source of biofeedback. Different modalities also display different responses over time. Finger temperature increases slowly as the user relaxes and lowers their sympathetic tone, whereas muscle tension and HRV respond relatively quickly to activities such as neck shrugging and unpaced breathing, respectively. Together, these varying modalities target different pathomechanisms and potentially embrace more users as individuals often master different forms of autoregulation. The use and combination of these three different modalities have been assessed thoroughly in preliminary studies using a progenitor version of the Cerebri system. First, a feasibility study was conducted to confirm the precision of using surface EMG measured from the skin overlying the upper trapezius fibers to measure neck muscle tension and using a photoplethysmograph placed on the index finger to measure peripheral skin temperature.7 Second, a usability study was conducted showing that it is possible to combine the modalities of neck muscle tension, finger temperature, and heart rate to tailor therapist-independent feedback to different users.8 Finally, HRV was included as the third parameter instead of simple heart rate measurement, as it was demonstrated in a study from the US,16 to be both feasible and acceptable for app-based biofeedback. The non-invasiveness of the Cerebri-system is advantageous in several ways. First, it enables the patients to safely perform the procedure themselves. Second, it means the risk profile is relatively low, with the most severe identified risks being electrical failure in the sensors or charger, skin contact-dependent allergic reactions from the sensor material, and risks related to information security and privacy. All the identified risks were met using an appropriate mitigation strategy. Third, a wider spectrum of patients qualifies for study inclusion; notably, women of childbearing potential who do not use highly effective contraceptives, as well as those who are pregnant or breastfeeding.\n\nThe BioCer trial is not blinded and employs a waitlist control. In a previous sham-controlled trial involving adolescents that tested an earlier version of the Cerebri system, the investigators encountered difficulties in developing a biofeedback sham that effectively imitated authentic biofeedback.9 False feedback was easily detected by the user, compromising the integrity of the blinding protocol. Conversely, attempts to distort the biofeedback signal by introducing minor “errors” resulted in a sham signal that was “too similar” to true biofeedback, potentially inducing a treatment effect. In addition to the practical challenge of developing an appropriate sham signal, there are significant ethical concerns associated with providing false biofeedback to participants. These concerns include the potential exacerbation of migraines. Indeed, in theory, introducing a false biofeedback signal could lead to mislearning, in which subjects inadvertently adapt incorrect or maladaptive responses based on what they believe to be true biofeedback.\n\nWe believe that the effectiveness of Cerebri depends on achieving adequate user satisfaction and adherence to ensure optimal results. However, a recurrent challenge for smartphone-based biofeedback is its low adherence. A pilot study in adolescents of a progenitor version of Cerebri reported poor adherence, with a mere 40% of planned biofeedback sessions completed during weeks 5-8 of the study.8 Similar adherence challenges have been reported for smartphone-based systems. In the feasibility study of the current Cerebri system proceeding this trial, biofeedback adherence was also low, declining from 80% in week 1-4 to 20% in week 9-12.11 Several usability and software issues were identified and addressed before trial initiation.\n\nTo improve the usability of the Cerebri mobile app, additional data were added to the diary and visualized in graphs to provide users with better tools to keep track of their headaches and to compare headache data with biofeedback data directly. In addition, the app content was revised and instructions were improved based on reported use errors or misinterpretations. Based on user insights, the app interface during the biofeedback sessions was tailored to ensure that the biofeedback was performed correctly. Furthermore, the algorithm for calculating the HRV score was modified to compensate for drifting biofeedback scores resulting from frequency leakage from the low-to high-frequency domains of the HR wave. Some drifting still occurs, but it is deemed acceptable as the users are considered unable to observe this.\n\n\nConclusion\n\nCerebri started as a research project in 2015, with a meta-analysis of the biofeedback literature on adolescents21 and concept development on how to enable remote biofeedback for headache patients. The project utilized existing technology in the market, and as the concept developed, custom-made sensors and a more advanced biofeedback app interface were developed over the course of several years. Clinical testing of feasibility and usability continued to show positive feedback from the patients8,10; hence, the project continued to validate the clinical effect of Cerebri. A feasibility study is important in validating the clinical study setup and defining the final iterations of the Cerebri user interface as the final preparation for this randomized controlled trial on Cerebri.\n\nThis study will be conducted in accordance with the principles of ISO 14155:2020 (Clinical Investigation of Medical Devices in Human Subjects), Good Clinical practice, and the Guidelines of the International Headache Society. The study was approved by the Regional Committees for Medical Research Ethics (REK, approval number 502734. Date of approval 2022-10-14) and the Norwegian Medical Product Agency (NOMA) (reference number 22/17254-5. Date of approval 2022-09-01) prior to initiation. Substantial protocol amendments will be reviewed by competent authorities before implementation. Data protection follows the GDPR. We adhere to the declaration of Helsinki.\n\nTo ensure that all subjects have similar opportunities to benefit from enrolment, participants randomized to the waitlist group were offered treatment following the conclusion of the waitlist period. Consent was collected by the investigator conducting the screening visit. Study information was sent in advance of visit 1 by email, scripted study information was provided orally, and any questions addressed during visit 1 prior to signing to ensure that consent was obtained in all cases. Consent was collected by a digital signature via the national electronic data entry application («Helsenorge»). Study personnel were prohibited from recruiting patients for whom they were directly responsible for treatment.\n\n\nAuthors information\n\nEAT, AO, and AS conceived of the study. AS initiated the study design. TWM has the role of project leader and coordination investigator. MS and TWM wrote the statistical analysis plan and MS will conduct the primary statistical analysis. ETVS and ACP prepared the manuscript for publication. ACP, LRØ, MHB, KGV, ESK, IKL, and TWM are study investigators. All authors contributed to the refinement of the study protocol and approved the final manuscript.", "appendix": "Data availability\n\nNo data is associated with this article.\n\nFigshare: SPIRIT checklist for ‘Biofeedback therapy using Cerebri for the prevention of migraine attacks in adults with episodic migraine (BioCer): A randomized, wait-list controlled trial. https://doi.org/10.6084/m9.figshare.25480723.v1.\n\nThe data are available under the terms of the Creative Commons Licence (CC BY 4.0).\n\n\nReferences\n\nStovner LJ, Nichols E, Steiner TJ, et al.: Headache in the Global Burden of Disease (GBD) Studies.Steiner TJ, Stovner LJ, editors. Societal Impact of Headache: Burden, Costs and Response. Cham: Springer International Publishing; pp. 105–125. Publisher Full Text\n\nLinde M, Gustavsson A, Stovner LJ, et al.: The cost of headache disorders in Europe: the Eurolight project. Eur. J. Neurol. 2012; 19: 703–711. PubMed Abstract \| Publisher Full Text\n\nHepp Z, Dodick DW, Varon SF, et al.: Persistence and switching patterns of oral migraine prophylactic medications among patients with chronic migraine: A retrospective claims analysis. Cephalalgia. 2017; 37: 470–485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilberstein SD: Practice parameter: Evidence-based guidelines for migraine headache (an evidence-based review) [RETIRED]. Neurology. 2000; 55: 754–762. PubMed Abstract \| Publisher Full Text\n\nAilani J, Burch RC, Robbins MS, et al.: The American Headache Society Consensus Statement: Update on integrating new migraine treatments into clinical practice. Headache. 2021; 61: 1021–1039. PubMed Abstract \| Publisher Full Text\n\nStubberud A, Linde M: Digital Technology and Mobile Health in Behavioral Migraine Therapy: a Narrative Review. Curr. Pain Headache Rep. 2018; 22: 66. PubMed Abstract \| Publisher Full Text\n\nStubberud A, Omland PM, Tronvik E, et al.: Wireless Surface Electromyography and Skin Temperature Sensors for Biofeedback Treatment of Headache: Validation Study with Stationary Control Equipment. JMIR Biomed. Eng. 2018; 3: e1. Publisher Full Text\n\nStubberud A, Tronvik E, Olsen A, et al.: Biofeedback Treatment App for Pediatric Migraine: Development and Usability Study. Headache. 2020; 60: 889–901. PubMed Abstract \| Publisher Full Text\n\nStubberud A, Linde M, Brenner E, et al.: Self-administered biofeedback treatment app for pediatric migraine: A randomized pilot study. Brain Behav. 2021; 11: e01974. Publisher Full Text\n\nIngvaldsen SH, Tronvik E, Brenner E, et al.: A Biofeedback App for Migraine: Development and Usability Study. JMIR Form Res. 2021; 5: e23229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoole AC, Winnberg I, Simpson MR, et al.: A Smartphone-Based Biofeedback Treatment for Episodic Migraine in Adults: A Feasibility Study of a 12-week Therapist-Independent Treatment. JMIR Human Factors. 2024. Submitted.\n\nDiener H-C, Tassorelli C, Dodick DW, et al.: Guidelines of the International Headache Society for controlled trials of preventive treatment of migraine attacks in episodic migraine in adults. Cephalalgia. 2020; 40: 1026–1044. PubMed Abstract \| Publisher Full Text\n\nMartin BC, Pathak DS, Sharfman MI, et al.: Validity and Reliability of the Migraine-Specific Quality of Life Questionnaire (MSQ Version 2.1). Headache. 2000; 40: 204–216. PubMed Abstract \| Publisher Full Text\n\nGuy W; National Institute of Mental Health (U.S.): Psychopharmacology Research Branch, Early Clinical Drug Evaluation Program. ECDEU assessment manual for psychopharmacology. Rev. Rockville, Md: U.S. Dept. of Health, Education, and Welfare, Public Health Service, Alcohol, Drug Abuse, and Mental Health Administration, National Institute of Mental Health, Psychopharmacology Research Branch, Division of Extramural Research Programs; 1976.\n\nNestoriuc Y, Martin A: Efficacy of biofeedback for migraine: A meta-analysis. Pain. 2007; 128: 111–127. Publisher Full Text\n\nMinen MT, Corner S, Berk T, et al.: Heartrate variability biofeedback for migraine using a smartphone application and sensor: A randomized controlled trial. Gen. Hosp. Psychiatry. 2021; 69: 41–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoser AE, Gibler RC, Ramsey RR, et al.: Digital headache self-management interventions for patients with a primary headache disorder: A systematic review of randomized controlled trials. Headache. 2022; 62: 1105–1119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNestoriuc Y, Martin A, Rief W, et al.: Biofeedback Treatment for Headache Disorders: A Comprehensive Efficacy Review. Appl. Psychophysiol. Biofeedback. 2008; 33: 125–140. PubMed Abstract \| Publisher Full Text\n\nLazaridou A, Paschali M, Bernstein C, et al.: sEMG Biofeedback for Episodic Migraines: A Pilot Randomized Clinical Trial. Appl. Psychophysiol. Biofeedback. Epub ahead of print 27 January 2024; 49: 271–279. PubMed Abstract \| Publisher Full Text\n\nCuneo A, Yang R, Zhou H, et al.: The Utility of a Novel, Combined Biofeedback-Virtual Reality Device as Add-on Treatment for Chronic Migraine: A Randomized Pilot Study. Clin. J. Pain. Epub ahead of print 2023; 39: 286–296. PubMed Abstract \| Publisher Full Text ,\n\nStubberud A, Varkey E, McCrory DC, et al.: Biofeedback as Prophylaxis for Pediatric Migraine: A Meta-analysis. Pediatrics. 2016; 138: e20160675. PubMed Abstract \| Publisher Full Text" }	[ { "id": "314897", "date": "30 Aug 2024", "name": "Aynur Özge", "expertise": [ "Reviewer Expertise Neurology", "Algology", "and Clinical Neurophysiology specialist. headache expert. An active member of the Pediatric SIG of IHS for more than 20 years." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript titled \"Biofeedback therapy using Cerebri for the prevention of migraine attacks in adults with episodic migraine (BioCer): a randomized, wait-list controlled trial – the study protocol\" presents a study protocol aimed at investigating the efficacy and safety of a novel biofeedback device, Cerebri, in preventing migraine attacks in adults with episodic migraines. The device integrates three biofeedback modalities: heart rate variability (HRV), peripheral skin temperature, and electromyography (EMG), allowing users to engage in home-based biofeedback therapy without the need for healthcare personnel. The study is an open-label, randomized, waitlist-controlled, multicenter trial with a primary endpoint of evaluating the change in the mean number of migraine days from baseline to the last 28-day period of the treatment phase. The study is significant as it addresses the accessibility of biofeedback therapy, offering a therapist-independent, home-based solution for migraine management. My Comprehensive Comments are as follows; Abstract:\nThe abstract is clear and concise, providing a good overview of the study's purpose, design, and significance. However, it would benefit from a brief mention of the study's limitations or challenges, such as the open-label design and the potential for low adherence, which is discussed in the manuscript.\nMethodology:\nThe methodology is thoroughly detailed, adhering to established clinical investigation standards. The inclusion of a baseline period as a screening phase is a strength, ensuring participants' eligibility is rigorously assessed. The open-label design, while necessary due to the nature of the intervention, introduces a potential bias that could be discussed more explicitly in the protocol. The lack of blinding is acknowledged, but further exploration of how this might affect the study's outcomes could enhance the robustness of the methodology section. The stratification and randomization process is well-explained, ensuring transparency in the study design.\nResults:\nAs this is a study protocol, the results section outlines the expected outcomes and statistical analyses planned. The use of a mixed logistic regression model is appropriate for the primary endpoint analysis, and the inclusion of both ITT and PP populations strengthens the study's rigor. The detailed plan for data management and statistical analysis, including handling protocol deviations, is a strong point of the manuscript.\nDiscussion:\nThe discussion section appropriately situates the study within the broader context of migraine treatment research. The manuscript effectively highlights the innovation of combining multiple biofeedback modalities and the potential impact of this approach on migraine management. The discussion on the challenges of adherence to smartphone-based biofeedback systems is well-founded and crucial to understanding the study's potential limitations. However, the discussion could be expanded to include potential strategies for improving adherence, which could be beneficial for future implementations of similar interventions.\nQuality of Tables and Figures:\nThe tables and figures are clear and well-organized, providing necessary visual support to the study protocol. The flowchart (Figure 1) is particularly useful in illustrating the study design and participant flow. The screenshots of the biofeedback treatment (Figures 3 and 4) effectively demonstrate how the Cerebri system operates, which adds clarity for readers who may not be familiar with biofeedback technology.\nThis manuscript presents a well-designed study protocol for evaluating a novel biofeedback device in migraine prevention. While the manuscript is strong overall, I suggest a minor revision to address a few areas: the abstract could briefly mention study limitations, the methodology section could include a more explicit discussion of the potential biases introduced by the open-label design, and the discussion could offer suggestions for improving adherence to the biofeedback regimen. Additionally, while the tables and figures are well-prepared, ensuring that they are not overly complex or repetitive will be important as the study progresses and results are reported.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-775
https://f1000research.com/articles/13-774/v1	08 Jul 24	{ "type": "Study Protocol", "title": "Digital well-being in children and youth: Protocol for a comprehensive systematic review of reviews on interventions of problematic digital technology use", "authors": [ "Jing Shi", "Jerome Jie Ming Tan", "Hwee En Ong", "Dahlia Aljuboori", "Saud Alomairah", "Michelle Colder Carras", "Johannes Thrul", "Jerome Jie Ming Tan", "Hwee En Ong", "Dahlia Aljuboori", "Saud Alomairah", "Michelle Colder Carras", "Johannes Thrul" ], "abstract": "Background Digital technologies proliferate in many people’s lives around the world with over 65% of these technology users being online. Children and youth are among the most prominent adopters of digital technologies in forms such as video gaming, social media, and online shopping. Problematic use of digital technologies can lead to poorer school/work performance, neglect of self-care skills, and comorbidities with other mental health issues. However, when used non-problematically, digital technology can also contribute to improving health and well-being. With the abundance of literature published, many reviews have sought to collate literature on treatment and interventions for children and youth with varying results. Thus, our proposed systematic review aims to synthesize current systematic reviews and meta-analyses on interventions and treatment of problematic digital technology use in children and youth (up to 25 years old).\n\nMethods As part of a three-paper series, a systematic search was completed in PsycINFO, Web of Science, and PubMed databases. Grey literature databases of the World Health Organization (IRIS database) and ClinicalTrials.gov were also searched. Furthermore, hand-searching of reference lists was also conducted. Title and abstract screening, followed by full-text screening, were completed by at least two independent reviewers. For this review, the extractions and the quality of selected reviews will be assessed using AMSTAR 2.0 by two authors independently and reviewed by two additional authors.\n\nResults Results will be presented in narrative and tabular form. The results of this study are expected to offer insights into the populations of children and youth studied, treatments/interventions provided, outcomes, results, limitations, and conclusions of literature from the past five years. Feasibility and generalizability of the reviews will also be discussed.\n\nConclusions Methodological strengths and weaknesses of reviewed studies will point to gaps in knowledge and can be used to inform future areas of policy and research.", "keywords": [ "digital wellbeing", "internet", "video games", "social media", "children", "youth", "intervention", "treatment" ], "content": "Introduction\n\nDigital technologies are a part of most people’s lives around the globe and around 65% of these technology users are connected to the Internet (Petrosyan, 2023). Digital technology is used as an umbrella term that involves the use of electronic devices (Dienlin & Johannes, 2020). Activities through digital technologies include Internet use, watching television, and the use of embedded apps within devices (Stiglic & Viner, 2019; Twenge & Campbell, 2018). Children and youth are using digital technologies that involve activities like video gaming, social connection apps, and online shopping (Adachi & Willoughby, 2017; Ibrahim et al., 2023; Richards & Caldwell, 2015; Shi et al., 2019). Other activities that historically did not involve digital technology have changed to digital form in recent years. Examples of these activities are reading on an e-reader (such as a Kobo or Kindle) or creating digital art. Newer forms of media involving digital technologies are listening to podcasts, engaging in AR/VR activities, participating in interactive shows from home, watching/interacting with live streamers, using AI (Partarakis & Zabulis, 2024), and blockchain activities.\n\nIn an increasingly digitized world where people spend a significant portion of their time using smartphones, computers, and other digital devices, the concept of digital well-being has become a critical consideration. Digital well-being is having a balanced and healthy relationship with technology (Vanden Abeele and Nguyen, 2022). It is also a measure of the impact that digital technologies have on what it means to live a “good” life (Burr & Floridi, 2020). Thus, digital well-being is a subjective individual experience that aims to strike a balance between the benefits and drawbacks that digital technologies offer.\n\nThis systematic review aims to synthesize existing literature reviews on any interventions and treatments for children and youth who use digital technologies problematically compared to any group on digital well-being outcomes. Thus, the objectives are to:\n\n1. Undertake a systematic search of the available systematic reviews and meta-analyses on clinical interventions and treatments of problematic digital technology use in children and youth.\n\n2. Synthesize the published reviews on clinical studies to inform clinical interventions and treatments.\n\n3. Evaluate the quality of the published reviews in terms of strengths, weaknesses, inconsistencies, and gaps in the evidence to inform future practice and research.\n\nThis study is part of a three-paper series of systematic and rapid reviews on digital well-being. The first review of this series on the prevention of problematic digital technology use is under peer review at the time of writing this protocol. A third review of empirical clinical studies will commence later. Thus, the methods of searching and screening articles that have already been completed will be outlined below. The methods used for extracting and evaluating the articles for this study will also be described in the sections below.\n\n\nMethods\n\nThis protocol was designed in accordance with the PRISMA-P statement for preferred reporting items for systematic reviews and meta-analyses protocol (Moher et al., 2015; Shamseer et al., 2015).\n\nWe aimed to find, assess, and synthesize all systematic reviews and reviews published containing clinical treatments or interventions for children and youth who use digital technologies. Articles were included if they were interventions for problematic use of digital technologies to improve well-being in children or youth. We excluded studies if they were prevention interventions as we have our first paper on prevention that is currently under peer review. The full list of inclusion and exclusion criteria for the selected studies are outlined in the sections below.\n\nInclusion criteria:\n\n• All languages (translation software will be used when the research team is unable to translate the text)\n\n• Systematic reviews and meta-analysis\n\n• Clinical treatment or intervention programs\n\n• Literature published in the last 5 years\n\n• Children, adolescents, and young adults (on average, up to 25 years old)\n\nAll interventions for problematic use of any form of digital technologies. Examples of such digital technologies include social media use, video gaming, Internet use (for productivity, leisure or information seeking), e-readers, and passive digital media consumption.\n\nExclusion criteria:\n\n• Conference publications, commentaries, case reports, and thesis dissertations\n\n• Digital gambling, loot boxes, betting, and all gambling interventions\n\n• Digital adult content pornography use\n\n• Average age of sample greater than 25 years and/or all participants are over 25 years old\n\n• Articles that focused on prevention interventions and/or prevention programs\n\n• Studies that are not interventions on digital well-being such as therapeutic uses of digital technologies (unless the aim was to improve digital well-being)\n\nThe databases used for this review include:\n\n1. PsycINFO (Database of Psychological Literature)\n\n2. Web of Science\n\n3. PubMed\n\n4. Grey literature databases\n\na. World Health Organization (IRIS database) and\n\nb. ClinicalTrials.gov\n\nHand-searching of reference lists of the selected review articles was also completed. In the event where a full-text was not accessible, we emailed the authors of the paper to request permission to access the full-text. The search terms in Box 1: Search Terms were used with Boolean operators. MeSH headings were used when appropriate. The search was started on March 3, 2023 and we anticipated to complete this review by the end of July 2024.\n\n(adolescents or teenagers or young adults or teen or youth or children)\n\nAND\n\n(Internet addiction disorder OR problematic online shopping OR impulsive online shopping OR excessive online shopping OR compulsive buying OR buying disorder OR Social media use disorder OR Problematic social media use OR Problematic social networking site use OR Social networking site addiction OR Social media addiction Social networking site dependence OR Pathological social networking site use OR Pathological social media use OR Compulsive social networking site use OR Compulsive social media use OR Excessive social networking site use OR Excessive social media use OR Problematic social networking site use OR Social networking site addiction OR Social media addiction OR Social networking site dependence OR Pathological social networking site use OR Pathological social media use OR Compulsive social networking site use OR Compulsive social media use OR Excessive social networking site use OR Excessive social media use OR screen time OR Gaming disorder OR Gaming addiction OR Heavy gaming OR Internet OR gaming disorder OR Addictive gaming OR addictive-problematic gaming OR internet gaming disorder OR addictive gaming OR problematic online shopping OR impulsive online shopping OR sedentary behavior OR excessive online shopping OR compulsive buying OR buying disorder OR Social media use disorder OR Problematic social media use OR Problematic social networking site use OR Social networking site addiction OR Social media addiction OR Social networking site dependence OR Pathological social networking site use OR Pathological social media use OR Compulsive social networking site use OR Compulsive social media use OR Excessive social networking site use OR Excessive social media use OR Internet addiction disorder OR Problematic social networking site use OR Social networking site addiction OR Social media addiction OR Social networking site dependence OR Pathological social networking site use OR Pathological social media use OR Compulsive social networking site use OR Compulsive social media use OR Excessive social networking site use OR Excessive social media use OR screen time OR Gaming disorder OR Gaming addiction OR Heavy gaming Internet OR gaming disorder OR Addictive gaming OR Impulsivity OR Behavioural addiction OR Behavioral addiction OR Pathological use)\n\nAND\n\n(Social media OR facebook OR tik-tok OR twitter OR Instagram OR tumblr OR Social Medium OR video game OR Computer Game OR facebook OR tumblr OR TikTok OR Pinterest OR youtube OR myspace OR wechat OR whatsapp OR reddit OR social network OR SNS OR 4chan OR online forum OR blog OR image sharing OR video-based OR image-based OR weibo OR social network OR social media OR social networking OR Gaming OR serious games OR educational games OR videogames OR therapeutic games OR active video games OR exergames OR dance games OR musical games OR gamification OR affective embodies pedagogical agent OR avatar OR prosocial video games OR collaborative games OR social online games OR brain training games OR commercial games OR exercise therapy OR biofeedback video games OR games as a distraction OR neuro-exercise game OR digital game program OR action video games OR media use OR advergames OR predatory monetization OR purchasing systems OR long-term cost of activity OR gamer-cognitions OR violant games OR violant media OR video game playing OR intensive video games OR spatial games OR virtual reality OR vr OR augmented reality OR ar OR mixed reality OR metaverse OR game development OR within-game creation OR Minecraft OR mine-craft OR modified commercial games OR online shopping)\n\nAND\n\n(wellbeing OR well-being OR well being OR digital balance OR digital detox OR digital wellbeing OR health technology OR screen time OR Interventions OR psychological interventions OR behavior modification OR addictive behaviors OR reality therapy OR Maladaptive cognitions OR cognitive-behavioral therapy OR craving behavioral intervention OR media regulations OR parenting style OR health behavior OR health behavior change OR digital balance OR Therapy OR Policy OR media regulations OR parenting style OR parenting interventions OR national interventions OR national preventions OR Intervention OR Treatment OR Therapy OR Therapeutics Program OR Programmes OR Behavioral modification)\n\nAll search results were imported to Covidence, a systematic review reference management software. Duplicates were removed automatically; additional duplicates were identified and manually screened out. Screening by title and abstract was conducted by a minimum of two authors independently. Following the screening of titles and abstracts, full texts were obtained for the remaining articles. Two authors reviewed the full texts against the inclusion criteria. The hand searching of reference lists were also completed by two members of the research team, independently. Additionally, to ensure accuracy, a third author reviewed at least 10% of all screened articles at each phase. Discrepancies at all three phases of screening and selection were discussed and resolved by consensus. Finally, searching, screening, and studies selected will be reported in a flow chart using the PRISMA Flow Diagram (Page et al., 2021).\n\nA standardised Microsoft Excel form will facilitate data extraction of the final selected studies. Two authors will extract all the studies while two additional authors will review the extractions to ensure accuracy. Should there be any uncertainties between the four authors, they will first discuss amongst themselves and then report the discussions to the first author. Authors of the review articles will be contacted in the case of any ambiguity.\n\nData to be extracted from the articles will include author, publication year, type of review, participants, digital technology/diagnoses, setting, aim of study, databases searched, date of last search update, number of included studies, treatment/interventions included, digital well-being outcomes assessed, results, author’s reported limitations, author’s conclusions, author’s future directions, and all criteria from the AMSTAR 2.0 checklist. A sample of the extraction table with headings and brief descriptions can be found in Table 1: Data Extraction.\n\nOur primary objective is to identify and categorize the types of treatments and interventions for children and youth who experience problematic digital use from literature reviews. Our secondary objective is to evaluate the evidence presented in literature reviews on this topic published within the past five years. We will identify potential strengths, limitations, inconsistencies, and gaps in the evidence.\n\nOur findings will be summarized into narrative form and extracted into a table. The narrative text will provide an explanation of our primary and secondary findings. Any overlaps of primary studies in the systematic reviews will be considered when drawing conclusions. Due to a mix of quantitative and narrative literature reviews that will be included in this systematic review, no meta-analysis is planned. However, we will discuss the applicability/feasibility and generalizability of the reviews on this population.\n\nThe strength of the body of evidence will be assessed by considering the following: 1) AMSTAR 2.0 assessments; 2) providing a clear research question with searches that capture the construct; 3) acknowledgment of review and methodological limitations.\n\nThe quality and risk of bias of the reviews will be assessed using the AMSTAR 2.0 checklist (Shea et al., 2017) by at least two authors independently for each review paper. An additional two authors will review assessments to ensure accuracy. Should there be any uncertainties between the four authors, it will be handled similarly to article extractions where they will first discuss amongst themselves and then report the discussions to the first author. Completed AMSTAR 2.0 checklists will be uploaded to our OSF website and a table of results will be presented in the final report.\n\nShould the protocol be amended, the date of each amendment will be provided with a description of the changes and the rationale for the changes on our OSF website. Dissemination of the systematic review results will be in the form of a peer-reviewed publication and/or a conference presentation.\n\n\nConclusions\n\nConsolidation of the literature on clinical interventions and treatments for children and youth who use digital technologies is needed. This study’s rigorous design along with risk-of-bias assessments of existing review literature is poised to provide valuable insights into the treatments and interventions for children and youths’ digital well-being. We hope that these insights will contribute significantly to advancements in the understanding of this field.\n\nNot applicable as this study is a systematic review of published literature and does not involve human/animal subjects.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nOpen Science Framework: PRISMA-P checklist for this article can be found at: https://osf.io/dg2ce/\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgements\n\nWe express our gratitude to Fatima Karkoub and Fasika Molla Abreha for their assistance during the initial stage of the larger project.\n\n\nReferences\n\nAdachi PJ, Willoughby T: The link between playing video games and positive youth outcomes. Child Dev. Perspect. 2017; 11(3): 202–206. Publisher Full Text\n\nBurr C, Floridi L: The ethics of digital well-being: A multidisciplinary perspective.Burr C, Floridi L, editors. Ethics of Digital Well-Being. Philosophical Studies Series. Cham: Springer; 2020; 140. . Publisher Full Text\n\nDienlin T, Johannes N: The impact of digital technology use on adolescent well-being. Dialogues Clin. Neurosci. 2020; 22(2): 135–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim NAN, Husseini F, Aisyah N, et al.: Online shopping behaviour in youth: A systematic review of the factors influencing online shopping in young adults. Int. J. Acad. Res. Bus. Soc. Sci. 2023; 13(2): 168–178. Publisher Full Text\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst. Rev. 2015; 4(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. BMJ. 2021; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPartarakis N, Zabulis X: A review of immersive technologies, knowledge representation, and AI for human-centered digital experiences. Electronics. 2024; 13(2): Article 2. Publisher Full Text\n\nPetrosyan A: Worldwide Digital Population 2023. Statista; 2023, October 25. Reference Source\n\nRichards D, Caldwell PHY: Impact of social media on the health of children and young people. J. Paediatr. Child Health. 2015; 51(2): 1152–1157. PubMed Abstract \| Publisher Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: Elaboration and explanation. BMJ. 2015; 349: g7647. 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\nShi J, Boak A, Mann R, et al.: Adolescent Problem Video Gaming in Urban and Non-urban Regions. Int. J. Ment. Heal. Addict. 2019; 17: 817–827. Publisher Full Text\n\nStiglic N, Viner RM: Effects of screentime on the health and well-being of children and adolescents: A systematic review of reviews. BMJ Open. 2019; 9(1): e023191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTwenge JM, Campbell WK: Associations between screen time and lower psychological well-being among children and adolescents: Evidence from a population-based study. Prev. Med. Rep. 2018; 12: 271–283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanden Abeele MMP, Nguyen MH: Digital well-being in an age of mobile connectivity: An introduction to the Special Issue. Mob. Media Commun. 2022; 10(2): 174–189. Publisher Full Text" }	[ { "id": "304359", "date": "29 Jul 2024", "name": "Veli-Matti Karhulahti", "expertise": [ "Reviewer Expertise Related to the present article: gaming disorder", "technology use", "meta-science." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review. I think this is my first protocol review; the publication format has not been very common in the fields related to my expertise. As I see it, the protocol format falls between preregistrations and Stage 1 RRs: it aims to describe the plan in more detail than preregistrations do (hence peer review), but not to the extent of RRs where sentence and word level detail is fixed in advance. Due to my regular involvement with editing and reviewing RRs, the below feedback may request “too much detail” in the present context, in which case the authors can naturally state that and ignore my point.\nIn general, the protocol is solid and will yield valuable results for related fields. Numerous reviews are being published continuously, and synthesizing that (meta) information is crucial for progress. I leave comments in a chronological order of their appearance in the manuscript.\nCriteria\nYou’re planning to include all languages and use LLMs to interpret the studies. In general it’s ok, but I’m curious how this is taken into consideration in the selected databases, screening, and search terms (and of course, study limitations). The translation software and its use could also be registered. Regardless, I’d encourage this to be further reflected on, especially considering the risks in presenting conclusions about cultures/languages of which a team may not have full expertise. The objectives of the study are presented at the end of the protocol. As a structural issue, I would prefer to have explicit RQs before the methods (and inclusion/exclusion criteria). This is because, as a reader, I find myself asking why certain criteria have and have not been selected. For example, why only search the last 5 years? Why exclude gambling and porn? This could be explained by presenting carefully crafted RQs preceding the criteria. The inclusion plan involves systematic reviews and meta analyses. Later on, however, on page 8 it is noted that narrative reviews will also be included. Narrative reviews (by most definitions) are not systematic, albeit there might be some hybrids. Defining ‘systematic’ (also for the coding process in the study) would be useful. Why conference proceedings are excluded? In my experience, many relevant reviews are published in HCI proceedings (HCI proceedings are among the most prestigious publication venues in that field). I don’t fully understand the last criterion; what kind of digital therapeutic intervention would not address wellbeing? All therapeutic interventions seem to have some wellbeing aspect. Will there be no backward and forward reference search? Especially if all languages are included, this might be a way to discover otherwise difficult to find reviews. EDIT: later in the protocol, there is a sentence saying that hand searching of reference lists were done; if this refers to back-forward search, please move the information to the search section.\nScreening and extraction\nWill coder-specific ratings and disagreements be reported in a supplement? Location of corresponding author will be extracted. I would prefer extracting wider team composition of authors and, if possible, looking at the regional/language differences in their included studies to shed light on the important cultural aspect. I would also be interested in the COIs, funders, and positionalities of authors in the reviews. This might be worth coding. I also notice that the protocol itself has no COIs; because the synthesis will be reported in narrative form, which allows a lot of rhetoric freedom, it might be worth adding positionality statements, if relevant ones exist -- some might say reviews like this shouldn’t have statements but I’ve made a case of there potentially being benefits [1].\nSynthesis\nThe synthesis and its reporting refer to primarily and secondary findings; primary being the types of treatments and the secondary being the assessment of evidence. It is not fully clear how the categorization of primary findings will occur. What method will be used for clustering? The secondary goal of assessment of evidence includes three listed elements. Again, I might be asking for too much detail, but considering that the epistemology of the review is somewhat on the positivist side, it would be important (in the final paper at least) to explain how each of the three listed elements are evaluated both respectively and in synthesis. I know this is a lot to ask; we recently tried to explain such process in detail and ended up with a rather complex plan [2]. Although I would not expect such detail here, the above planning taught me how much freedom there is in any synthesis process and we as researchers have a lot of power in it.\nBecause I've never reviewed a protocol before, I had a chat with my colleague Marcel (we work in the same project daily) about the manuscript and he, as an expert of systematic metas/reviews, got so curious that he offered to write a co-review. Hopefully you find his additional feedback helpful. (A lot of it overlaps with what I wrote above, but we decided not to edit our reviews for transparency, as we wrote them independently.)\nMarcel\nINTRODUCTION\n1) It appears that the Introduction is currently quite broad in scope. In my opinion, it would be more appropriate to focus the Introduction on defining and clarifying the main construct of this review, which is the problematic use of digital technologies. I was struggling to find its definition. The keywords used in the search string partially gave me a little hint.\nThe Inclusion criteria list some examples, such as Internet use (for productivity, leisure, or information seeking) and e-readers, but e-readers, for instance, are not included in the search string. Based on the keywords used in the search strings, it seems that 'problematic digital technology use' is limited to internet addiction, online shopping, social media addiction, and gaming addiction. May I inquire why constructs such as smartphone addiction, streaming addiction, excessive online dating app use, crypto trading addiction, excessive use of virtual reality environments, excessive use of fitness tracking or health apps, excessive use of smart home devices or IoT were not included? Similarly, could you explain why Digital adult content pornography use was excluded?\n2) It would be beneficial to specify more clearly what types of interventions this review covers. Interventions focused on what construct? Later in the text, it's mentioned that these are 'interventions for problematic use of digital technologies to improve well-being'. What did the authors mean by the term 'well-being'? (this construct is extremely vague and broad). Does it also relate to other related constructs?\nMETHODS\nEligibility criteria\n1) The eligibility section mentions 'all systematic reviews and reviews', but the inclusion criteria only list systematic reviews. Are the authors including all types of reviews or only systematic ones?\nInclusion criteria\n2) The authors state that they want to include all languages. How do they intend to achieve this if their keywords are only in English? 3) I'd appreciate an explanation for the inclusion of only 'Literature published in the last 5 years.' What is the rationale behind this decision? 4) Have the authors considered expanding the search in Grey literature databases to include preprints and Google Scholar? 5) The authors state that 'Additionally, to ensure accuracy, a third author reviewed at least 10% of all screened articles at each phase.' However, I couldn't find any mention of the level of agreement between reviewers. What measures are in place in case of low agreement? How will accuracy be ensured? 6) Data extraction: The extracted data should be explained in detail. Many descriptions are unclear about what should be coded and how, for example the code: 'setting'. It would be beneficial if the coding sheet had already undergone a pilot coding phase, and individual codes, their explanations, and instructions had been tested. [VMK note: as an example of preregistered coding, the materials of the synthesis that I mentioned earlier (2) can be found here: https://osf.io/shprf/] 7) How will the authors 'identify and categorize the types of treatments and interventions'? Will this be based on some existing classifications or done inductively?\nOutcomes and prioritization\n8) I find the text lacking an explanation of what exactly the authors mean by 'inconsistencies and gaps in the evidence.' How do they intend to identify these? Similarly, it wasn't clear to me how the 'acknowledgment of review and methodological limitations' will contribute to the assessment of the strength of the body of evidence. Do the authors aim to merely check for the presence/absence of limitations, or do they intend to evaluate the nature of the reported limitations as well? What about cases where original review authors don't mention any limitations, but the review objectively contains several? Do the authors plan to analyze the limitations of each included review? 9) The authors mention that they will synthesize both meta-analyses and reviews. How do they plan to combine their results, given their qualitatively different nature? What is their plan or procedure for doing this?\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/13-774
https://f1000research.com/articles/13-770/v1	08 Jul 24	{ "type": "Research Article", "title": "Laryngeal nerves in the cadaveric neck and its relevance to neck surgery- A descriptive study", "authors": [ "Prameela M Dass", "Ashwin R Rai", "Mangala M Pai", "Muralimanju B V", "Ganesh K Chettiar", "Rajalakshmi Rai", "Prameela M Dass", "Ashwin R Rai", "Mangala M Pai", "Muralimanju B V", "Ganesh K Chettiar" ], "abstract": "Background Because of its close relationship with vessels that supply the thyroid gland, it is necessary to understand and document the course of the laryngeal nerves to prevent accidental damage. Otherwise it can lead to weakness in vocal cord function if ligated along with the arteries during thyroidectomy or other surgical procedures. The aim of our study was to examine and document the origin, relationship, and branching patterns of the laryngeal nerves on the side of the neck.\n\nMethod The present cross sectional anatomical study was conducted on 64 dissected necks of formalin-fixed cadavers that were utilized for undergraduate teaching purposes. Different parameters were measured in the neck along with the relationship between the laryngeal nerves and their accompanying arteries.\n\nResults We observed usual and unusual course of laryngeal nerves along with some variations. Some of the novel findings were an internal laryngeal nerve piercing the thyroid cartilage lamina and an external laryngeal nerve communicating with the superior cervical ganglion.\n\nConclusion The present study observed anatomical variations in the course of the laryngeal nerves, which are relevant for clinical practice. The potential risks of damage to these laryngeal nerves during various surgical procedures make it necessary to review and compile its relationship with the surrounding structures.", "keywords": [ "Recurrent laryngeal nerve", "external laryngeal nerve", "internal laryngeal nerve", "thyroid surgery", "neck surgery" ], "content": "Introduction\n\nInnervation of the larynx plays an important role in the respiration, vocalization, coughing, deglutition, and vomiting reflexes. It is by superior (SLN) and recurrent laryngeal (RLN) branches of Vagus nerve (VN) (Standring, 2014).1 The SLN is further divided into the external and internal laryngeal nerves (ELN and ILN, respectively) before supplying the larynx. The ILN is essentially sensory and supplies the mucosa of the supraglottic part of the larynx. Along its course, it pierces the thyrohyoid membrane (TM) and is accompanied by superior laryngeal artery (SLA). The ELN, which mainly contains the motor component, descends along the superior thyroid artery (STA) to supply the cricothyroid muscle (CTM). RLN containing motor and sensory components supply all muscles of the larynx, except the CTM, and innervate the laryngeal mucosa below the vocal cord. In the neck it lies in the groove between the trachea and the esophagus before entering the larynx (Standring, 2014).1 Owing to their proximity to the vessels supplying the thyroid gland, these laryngeal nerves are vulnerable to injuries, generally during thyroid surgeries. SLN injury is subtle, usually unnoticed, and challenging to detect (Teitelbaum et al., 1995).2 Generally, SLN injury is confused with subluxation of the arytenoid, even after thorough evaluation (Schroeder et al., 2003).3 Consequently, laryngeal endoscopy and electromyography are required to diagnose SLN injuries (Teitelbaum et al., 1995).2 The ILN is vulnerable to injury during neck surgeries using an anterior or anterolateral approach (AbuRahma et al., 2000, Melamed et al., 2002).4,5 Iatrogenic injuries to the ELN occur during ligation of the STA for thyroidectomy and cause postoperative complications such as hoarse and weak voice (Sakorafas et al., 2012; Cernea et al., 1992).6,7 The variable relationship between the RLN and inferior thyroid artery (ITA) near the base of the thyroid gland makes it vulnerable to accidental injuries during thyroidectomy. The risk of permanent RLN palsy is 0.3% -3% and transient palsy is 3%-8% (Hayward et al., 2013).8 RLN injury may lead to laryngeal muscle paralysis, with symptoms ranging from hoarseness of voice in unilateral lesions to stridor and laryngeal obstruction in bilateral damage (Erbil et al., 2007; Jeannon et al., 2009; Kahky et al., 1993; Tang et al., 2012).9–12 Even if intraoperative neuromonitoring using the RLN technique is used to prevent RLN injury, evidence indicates that it is as effective as visualization of the RLN during dissection and is not greater than it (Hayward NJ et al., 2013; Calo et al., 2013; Phelan et al., 2013).8,13,14 The potential risks of damage to these laryngeal nerves during various surgical procedures necessitate precise documentation of its topographic relations.\n\nThe literature review did not reveal enough studies with respect to the laryngeal nerves in cadaveric samples, particularly from our sample population. This was a rationale to perform this research and the aim of our study was to examine and document the origin, relationship, and branching patterns of the laryngeal nerves on the side of the neck. The specific objective was to document the anatomical variations in the course of laryngeal nerves. It is hypothesized that the Vagus nerve and branches are vulnerable injury because of its wandering course and complex branching pattern.\n\n\nMethod\n\nThis is an observational cross-sectional, institution based study conducted on 64 dissected neck of formalin fixed cadavers, which were utilized for undergraduate teaching purpose, in the department of Anatomy of our institution from 2019 to 2023. The convenient sampling method was considered based on the availability of specimens at our department in this study period. The source of cadavers were from donated bodies, the body donor had given the written consent before donating the body. The study protocol is approved by Institutional Ethics Committee of Kasturba Medical College, Mangalore (ECR/541/Inst/KA/2014/RR-17) on 21st August 2019 (IEC KMC MLR 08-19/355).\n\nOnly adult cadavers were included for the study and if there were any pathological changes, the same were excluded. Since there were very few female cadavers in our department, the gender based comparison was not performed.\n\nAll measurements were in millimeters, measured with the help of a scale, thread, and caliper. The measurements were performed by the same person to prevent inter-observer bias. The measurements were also performed thrice, and the average was taken to prevent intra-observer bias. The following parameters were observed bilaterally in these specimens:\n\n1. Length of ILN from its origin till it pierces the TM\n\n2. Vertical distance between ILN and the upper border of thyroid lamina\n\n3. Relation between ILN and SLA\n\n4. Branches of ILN before piercing the TM\n\n5. Length of ELN from its origin to its termination on CTM\n\n6. Relation of ELN with inferior constrictor muscle (ICM)\n\n7. Vertical distance between ELN and apex of thyroid gland (AT)\n\n8. Branches of ELN to AT\n\n9. Relations of RLN with trachea and oesophagus\n\n10. Relations of RLN with ITA\n\n11. Distance between the base of thyroid lobe and crossing of RLN and ITA\n\n12. Number of branches of RLN before it enters the larynx\n\n13. Distance between entry of RLN into larynx and CTJ\n\nThe length of the ILN and ELN were measured using a measuring scale and thread. The other distances in the study were measured using a digital Vernier caliper. Since this is a descriptive study, the variations in the course and relation of the ELN and RLN are expressed as percentages.\n\nThe protocol of this study is available online at https://www.protocols.io/file/piy5cbrd7.docx.\n\n\nResult\n\nMeasurement of the length of the ILN, distance between the ILN from the upper border of the thyroid lamina, Length of ELN, distance between the ELN and AT, distance between the base of the thyroid lobe and crossing of the RLN and ITA, and the distance between the entry of the RLN into the larynx and the CTJ are compiled in Table 1.\n\nRelation of ILN & superior laryngeal artery (SLA): In the majority of cases, ILN was found posterior to the SLA, followed by cranial and caudal relations, as depicted in Table 2 (Fig. 1a,b, and c). Branches of ILN: in the majority of the specimens, ILN was single and did not give any branch before piercing the TM, as shown in Table 3 (Fig. 2a, b, c & d).\n\n1a. ILN is posterior to the SLA: 1b. ILN is cranial to the SLA: 1c. SLA is caudal to the ILN.\n\nELN- external laryngeal nerve; CCA- common carotid artery; ICA- Internal carotid artery; ECA- external carotid artery. STA- superior thyroid artery; SG- Submandibular gland; TG- Thyroid gland; TM- Thyrohyoid membrane; TC- Thyroid cartilage, HN-hypoglossal nerve.\n\n2a: ILN without branches; 2b: ILN giving two branches one of the branch (*) supplying ICM;\n\n2c: ILN giving three branches; 2d: ILN giving multiple branches.\n\n1,2-2 branches of ILN, ELN- External laryngeal nerve; CCA- common carotid artery; ECA- external carotid artery; STA- Superior thyroid artery; SG- Submandibular gland; ICM- Inferior constrictor muscle; TG- Thyroid gland; 1,2,3- 3 branches of ILN; T-thyroid cartilage; TM- Thyrohyoid membrane; 1,2,3,4,5-5 branches of ILN.\n\nOn the right side of one cadaver, the ILN pierced the lamina of the thyroid cartilage, 4 mm below its upper border (Fig. 3).\n\nILN- Internal laryngeal nerve, T- Thyroid lamina.\n\nELN- external laryngeal nerve, STA- Superior thyroid artery, CCA- Common carotid artery, IJV- Internal jugular vein, PD- Posterior belly of the digastric.\n\nBranches of ELN: In all specimens, it supplied the cricothyroid muscle (CTM). However, in one left-sided specimen, the ELN communicated with a branch of the superior cervical sympathetic ganglion. (Fig. 4)\n\nILN- internal laryngeal nerve; C- Communication of ELN with S, CCA- Common carotid artery (cut and reflected up); V- Vagus nerve.\n\nRelation between ELN & ICM:\n\n• In 90.6% of specimens, ELN was superficial to ICM,\n\n• In 6.25% it was piercing the ICM near its lower border,\n\n• In 2 specimens it pierced the ICM near its middle part\n\nDistance between ELN & apex of thyroid gland: Fig. 5a & b.\n\n5a: ELN is close to STA at apex of TG on right side of neck (< 1cm);\n\n5b: ELN is very close to STA at apex of TG on right side of neck ( >1cm);\n\n5c: ELN giving branches to apex of thyroid gland lobe on right side of neck.\n\nSTA- superior thyroid artery, STV- Superior thyroid vein, TG- Thyroid gland, CCA- Common carotid artery, ECA- External carotid artery, ICA- Internal carotid artery, SG- Submandibular gland, V- vagus nerve, IJV- Internal jugular vein.\n\nIn the majority of specimens (90.6%), the distance between the ELN and AT was less than 1 cm (average 5.22 mm). These distances are tabulated in Table 4.\n\nIn 68.75% of specimens, the RLN was in the tracheoesophageal groove, whereas in 31.25%, it was lateral to the trachea.\n\nRelation between RLN & inferior thyroid artery (ITA):\n\nIn the majority of specimens, the RLN was posterior to the loop of the ITA, as shown in Table 5 (Fig. 6a, b, c and d).\n\n6a: RLN posterior to Inferior thyroid artery.\n\n6b: RLN anterior to inferior thyroid artery and also shows 4 branches of RLN by red stars.\n\n6c: RLN passing in between the branches of inferior thyroid artery on left side of neck.\n\n6d: ITA passing in between the branches of RLN on right side of neck.\n\nTG- thyroid gland; T- Trachea, CCA- Common carotid artery; V-vagus nerve, TG- Thyroid gland, SCM- Sternocleidomastoid.\n\nBranches of RLN: In the majority of specimens, RLN provided three branches, as tabulated in Table 6 (Fig. 7a, b and c).\n\n7a: RLN without any branches on right side of neck.\n\n7b: RLN giving 2 branches on the left side of neck indicated by red stars.\n\n7c: RLN giving 3 branches on the right side of neck indicated by black arrows.\n\nITA- inferior thyroid artery; TG- Thyroid gland; T- Trachea, CCA- Common carotid artery; V-Vagus nerve; SCM- Sternocleidomastoid muscle.\n\n\nDiscussion\n\nAvoiding nerve or vascular injury is the basic principle behind the success of any surgery. Familiarity with the usual length of the laryngeal nerves, their relationship with neighboring structures, and accompanying arteries will facilitate successful surgeries such as thyroidectomy, carotid endarterectomy, or cervical spine surgery.\n\nThe knowledge of anatomical localization of ILN is a prerequisite for surgeries involving sensory denervation of the larynx for odynophagia or neuralgia of superior larynx, supraglottic laryngectomy and laryngeal transplantation (Cernea et al., 1992; Yanagisawa et al., 1970; Schmidt et al., 1981; Strome et al., 2001)7,15–17 The average length of ILN in previous studies was 44.9 mm (Furlan et al., 2003)18 and 57.2 mm (Kiray et al., 2006).19 The mean length of ILN was 33.8 mm in our study (Table 1).\n\nILN and its relation with Thyrohyoid membrane\n\nThe identification of the area of the TM pierced by the ILN is crucial in the dissection of the neck for hypopharyngeal squamous cell carcinoma because any injury to this nerve may prove to be fatal due to loss of cough reflex (Sun et al., 2022).20 We observed a distance of 2 mm to 12 mm (0.2-1.2 cm), between the ILN from the point of penetrating the TM to the upper border of the thyroid lamina, with an average of 6.33 mm (0.63 cm). Paraskevas et al. (2012) noticed that this distance ranged between 0-1.8 cm, but in 87.5% of cases, it ranged from 0.1 to 1.2 cm.21 They described the 0-1.8 cm range as the ‘wide zone’ and 0.1 to 1.2 cm as the ‘danger zone’ between TM penetration of ILN and thyroid cartilage. Familiarity with the ‘danger zone’ and any deviation from the usual pattern can be helpful in preserving ILN during surgical procedures in this region. Guven et al., 2021 have observed this distance ranging from 6-16 mm (0.6-1.6 cm) with the mean of 12 ±2.61 mm (1.2±0.26 cm) in patients undergoing laryngectomy.22\n\nRelation between SLA and ILN\n\nAlthough a varied relationship between ILN and SLA was observed in our study, in majority of the specimens, the ILN was posterior to the SLA before piercing the TM (51.56%) ((Table 2; Fig. 1a). It was above (cranial) the SLA in 29 specimens (45.31%) (Fig. 1b) and below (caudal) the SLA in two specimens (3.1%) (Fig. 1c). In contrast, Kiray et al. (2006) observed SLA crossing the ILN and piercing the TM above the ILN in 75% and below it in 25% of cases.19 Furlan et al. (2003) reported an ILN medial to the SLA in 89% and distal to the SLA in 11%.18 Paraskevas et al. (2012 have observed SLA parallel caudal and anterior to the ILN in 88.89% and superior and anterior to the ILN in 11.11% of cases.21\n\nBranching pattern of ILN (Table 3)\n\nThe branching pattern of the ILN before it pierces the TM has been studied by several authors. Stephen et al. (1999) reported that ILN branched before piercing TM in 16% and after penetration in 84% of cases,23 whereas according to Kiray et al., it was 37.5% and 62.5%, respectively.19 In a study by Guven et al. (2021), ILN was divided into three branches in 58.62% of cases and two branches in 41.37% of cases.22 Paraskevas et al. (2012) reported three branches of ILN before it pierces the TM in 72.22% of cases and two branches in 27.78% of cases.21 In our study, ILN did not give any branch before piercing the TM in 84.37% (Fig. 2a), in 10.93% it was divided into two branches (Fig. 2b), in 3.1% of the three branches (Fig. 2c), and in only one right-side specimen, it was divided into multiple branches (Fig. 2d). Awareness of the branching pattern of the ILN is important during surgical procedures of the larynx, such as laryngectomy. Even if a single branch of the ILN is removed during these procedures, it may cause significant postoperative complications; therefore, re-innervation of these branches is crucial in restoring the reflex function of the larynx. Additionally, we observed a rare variation in ILN piercing the lamina of the thyroid cartilage in one right-sided specimen (Fig. 3). This variant course may result in some unexplained complaints due to nerve compression, leading to misinterpretation by the clinician if he/she is unaware of such a variation.\n\nThe principles of thyroidectomy are based on the identification and preservation of the ELN, as injury to the ELN is detrimental to the patient by producing a weak and hoarse voice. The close relationship between the ELN and the upper pole of the thyroid gland makes nerves vulnerable to injury during thyroid surgery. The lower the deviation of the nerve from the STA, the higher is the risk of injury during dissection (Potenza et al., 2017).24\n\nIn our study, the mean length of the ELN was 58 mm (Table 1), whereas Furlan et al. (2003) reported a mean length of 62.6 mm,18 Thomassin et al., (1982)25 as 65 mm, and Kambic et al., (1984) as 80 mm.26 The course of the ELN has been studied by several authors (Cernea et al., 1995; Kierner et al., 1998; Cha et al., 2017; Friedman et al., 2002).27–30 Cernea et al. (1995) were the first to describe a classification of ELN with respect to its relationship with the STA.27 Accordingly, they classified 3 types of ELN wherein, type 1 crosses STA more than 1 cm above the apex of the thyroid gland (AT), type 2a crosses STA less than 1 cm above the AT and Type 2b crosses STA under AT cover. They mentioned that type 2b has a higher risk of damage than type 1 because, in this type, ELN is very close to STA. Kierner et al., (1998) revised nomenclature of Cernea et al., (1995) and described four types of ELN, where type 1 (42%), type 2 (30%) and type 3 (14%) are the same as type 1, type 2a and type 2b, respectively of Cernea et al., classification.28 In type 4, the ELN descends posterior to the STA and crosses its branches just above the AT. In our study commonest finding of ELN was type 2 (< 1 cm) of Kierner et al, in 82.81% (Fig 5a) followed by type 1 (> 1 cm) in 9.37% and type 3 in 7.81% (Table 4). We did not encounter Kierner et al. type 4 in our study. Cha et al. (2017) found ELN <1 cm distance from AT in 51.7%, >1 cm distance from AT in 27.6%, and under AT in 20.7%.29\n\nELN and inferior constrictor (ICM) & cricothyroid muscle (CTM)\n\nFriedman et al. (2002) described three types of ELN in relation to ICM, CTM and STA.30 In type 1, ELN runs superficial to ICM throughout, descending with STA until its termination in CTM. In type 2, the ELN penetrates the ICM in its lower part, whereas in type 3, the ELN penetrates the ICM in its upper part. Type 2 and type 3 ELN of Friedman et al. (2002) are difficult to identify during thyroid surgery as they are deep to the ICM but are safer than type 1, as type 1 is more prone to injury. We observed type 1 of Friedman et al., in 90.6% of the specimen, type 2 in 6.26%, and type 3 in 3.1% of the specimens in our study. Few authors have reported these as unidentified ELN (Jonas et al., 2000; Lore et al., 1998).31,32 In such situations, the ELN can be identified by nerve stimulation at the junction of the ICM and CTM (Thomassin JM., 1982).25 Additionally, we observed a rare variation wherein the ELN communicates with the superior cervical ganglion (Fig. 4). Such variation might lead to disturbances in sympathetic function if the ELN is injured during thyroid or other neck surgeries.\n\nThe RLN is closely related to the ITA near the base of the thyroid gland, and inadvertent injury to the RLN may lead to hoarseness of voice and cough if unilateral. Bilateral injury may cause dyspnea or life-threatening laryngeal obstruction (Jiang et al., 2014).33\n\nRLN and trachea-oesophageal groove\n\nThe course of RLN makes it vulnerable to injuries during certain surgical interventions in the neck (Culp et al., 2023).34 This can include surgeries involving the thyroid or parathyroid gland (Joliat et al., 2017),35 laryngeal malignancies (Benninger et al., 1998)36 or endotracheal intubation (Norris et al., 2011).37 We found RLN in the tracheoesophageal groove in 68.75% and lateral to the trachea in 31.25% of the patients. Sailaja (2016) reported RLN lateral to the trachea on 27 (15 right, 12 left) sides and in the tracheoesophageal groove on 23 sides (10 right, 13 left).38 Shao et al., (2016) have also reported that the right RLN is more anterolateral in position than the left RLN.39 Asgharpour et al., (2012) have seen the RLN anterior to the tracheoesophageal groove in 41.6%, in the groove in 33%, and posterior to the groove in 24.5%.40 They also found that the right RLN is more anterior than the left side. They opine that the right RLN is more exposed than the left. This may be due to differences in the embryological origin.\n\nRLN and relation with inferior thyroid artery (ITA):\n\nSurgical injuries are the most common cause of RLN palsy, accounting for 11 to 32% (Titche, 1976).41 Surgeries which can cause injury to the RLN are thyroidectomy, parathyroidectomy, esophagectomy, tracheoplasty, mediastinoscopy, correction of patent ductus arteriosus, etc. (Titche, 1976; Friedrich et al., 1998).41,42 Thyroidectomy is the most common cause of RLN injury, accounting for approximately 5 to 14%, according to various studies (Hayward et al., 2013; Friedrich et al., 1998, Zakaria et al., 2011).8,42,43 This can occur when a branch of the ITA is accidentally injured during the procedure. In most specimens (50%), the RLN was posterior to the ITA compared to other positions (Table 5; Fig. 6a, b, c, d) in our study. This is in accordance with the results of Tang et al. (2012) and Sailaja (2016) (86.25% and 56%, respectively).12,38 On the contrary, Campos et al. (2000) observed RLN behind the ITA in a comparatively lesser number of specimens.44 However, they observed RLN passing between the branches of ITA in the majority of the cases they studied (≈50%). When the RLN passes between the branches of the ITA, as shown in Fig. 6c, it makes it more vulnerable to injury during dissection for surgical procedures. Few researchers have opinioned that RLN injury is more common when the nerve is anterior or between the branches of the ITA than posterior to it (Skandalakis et al., 1976; Steurer et al., 2002).45,46 Surgeons should be aware of these types of variable relations between the RLN and ITA to avoid nerve damage.\n\nBranching pattern of RLN\n\nThe number of branches from the RLN before it pierces the muscles of the larynx is clinically significant because the branches may entangle the ITA, place the nerve at risk, and paralyze the laryngeal muscles (Shao et al., 2016).39 In our study, a varied number of branches of the RLN before piercing the larynx was found, with a maximum of 64% (Table 6; Fig. 7a, b & c). Sailaja (2016) revealed that the number of branches of the RLN was 2 (75%), 3 (9%), more than 3 in 8%, and 0 in 8%.38 Common pattern of extralaryngeal branching of the RLN is bifurcation with an incidence of 51%, and trifurcation or multiple branches have been reported by a few authors (Shao et al., 2016; Cakir et al., 2006; Henry et al., 2016).39,47,48 This type of branching of RLN before its entry into the larynx is the common cause for surgical morbidity in thyroid surgery (Shao et al., 2016).39 Other than ITA, other reliable landmarks used for the identification of the RLN are distance between the entrance of RLN to the larynx and inferior cornu of the thyroid cartilage or inferior tubercle of the thyroid cartilage or anterior part of cricoid cartilage (Cakir et al., 2006).47 We have found the mean distance between the cricothyroid joint and the entrance of RLN into the larynx as 19.5 mm with the range of 13 mm and 30 mm. The present study has few limitations like effect of formalin on the dimensions of the nerves and gender based comparison, which was not performed.\n\n\nConclusion\n\nIn the present study, we recorded the usual course of laryngeal nerves along with some rare variations, such as ILN piercing the thyroid cartilage lamina or multiple branches of the RLN that lie in close proximity to the branch of the ITA. The compiled data may aid in the success of surgeries in the neck, in and around the laryngeal nerves by minimizing the risk of their accidental damage. The findings of this study can be considered as a morphological data base of the laryngeal nerves, for our sample population.\n\nThis study was conducted on 64 dissected neck of formalin fixed cadavers, which were utilized for undergraduate teaching purpose, in the department of Anatomy of our institution. The study protocol is approved by Institutional Ethics Committee of Kasturba Medical College, Mangalore (ECR/541/Inst/KA/2014/RR-17) on 21st August 2019 (IEC KMC MLR 08-19/355).\n\nConsent form was waived by the Institutional Ethics committee, as it is a cadaveric study. However the body donors had given written consent before donating the body to our department.", "appendix": "Data Availability\n\nFigshare: Morphometry of laryngeal nerves in cadavers, https://doi.org/10.6084/m9.figshare.25002848. 49\n\nThis project contains the following underlying data:\n\n• Dataset\n\nData is under license CC0\n\n\nReferences\n\nStandring S: Gray's Anatomy- The Anatomical Basis of Clinical Practice. 40th ed. London: Churchill-Livingstone; 2014; 459.\n\nTeitelbaum BJ, Wenig BL: Superior laryngeal nerve injury from thyroid surgery. Head Neck. 1995; 17(1): 36–40. Publisher Full Text\n\nSchroeder U, Motzko M, Wittekndt C, et al.: Hoarseness after laryngeal blunt trauma: A differential diagnosis between an injury to the external branch of superior laryngeal nerve and an arytenoid subluxation. A case report and literature review. European Archives of Oto-Rhino- Laryngology. 2003; 260(6): 304–307. PubMed Abstract \| Publisher Full Text\n\nAbuRahma AF, Choueiri MA: Cranial and cervical nerve injuries after repeat carotid endarterectomy. J. Vasc. Surg. 2000; 32: 649–654. PubMed Abstract \| Publisher Full Text\n\nMelamed H, Harris MB, Awasthi D: Anatomic considerations of superior laryngeal nerve during anterior cervical spine procedures. Spine. 2002; 27: E83–E86. PubMed Abstract \| Publisher Full Text\n\nSakorafas GH, Kokoropoulos P, Lappas C, et al.: External branch of the superior laryngeal nerve: applied surgical anatomy and implications in thyroid surgery. Am. Surg. 2012; 78: 986–991. PubMed Abstract \| Publisher Full Text\n\nCernea CR, Ferraz AR, Nishio S, et al.: Surgical anatomy of the external branch of the superior laryngeal nerve. Head Neck. 1992; 14: 380–383. Publisher Full Text\n\nHayward NJ, Grodski S, Yeung M, et al.: Recurrent laryngeal nerve injury in thyroid surgery: A review. ANZ J. Surg. 2013; 83(1-2): 15–21. Publisher Full Text\n\nErbil Y, Barbaros U, Issever H, et al.: Predictive factors for recurrent laryngeal nerve palsy and hypoparathyroidism after thyroid surgery. Clin. Otolaryngol. 2007; 32: 32–37. PubMed Abstract \| Publisher Full Text\n\nJeannon JP, Orabi AA, Bruch GA, et al.: Diagnosis of recurrent laryngeal nerve palsy after thyroidectomy: a systematic review. Int. J. Clin. Pract. 2009; 63: 624–629. PubMed Abstract \| Publisher Full Text\n\nKahky MP, Weber RS: Complications of surgery of the thyroid and parathyroid glands. Surg. Clin. North Am. 1993; 73: 307–321. Publisher Full Text\n\nTang WJ, Sun SQ, Wang XL, et al.: An applied anatomical study on the recurrent laryngeal nerve and inferior thyroid artery. Surg. Radiol. Anat. 2012; 34: 325–332. PubMed Abstract \| Publisher Full Text\n\nCalo PG, Pisano G, Medas F, et al.: Intraoperative recurrent laryngeal nerve monitoring in thyroid surgery. Is it really useful? Clin. Ter. 2013; e193–e198.\n\nPhelan E, Schneider R, Lorenz K, et al.: Continuous vagal IONM prevents RLN paralysis by revealing initial EMG changes of impeding neuropraxic injury: A prospective, multicentric study. The larygoscope. 2013; 124(6): 1–8.\n\nYanagisawa E, Christmas DA, Wilson GL: Superior laryngeal nerve section for odynophagia. Arch. Otolaryngol. 1970; 91: 387–388. PubMed Abstract \| Publisher Full Text\n\nSchmidt D, Strutz I: Superior laryngeal neuralgia. J. Neurol. 1981; 225: 223–225. Publisher Full Text\n\nStrome M, Stein J, Esclamado R, et al.: Laryngeal transplantation and 40 months follow-up. N. Engl. J. Med. 2001; 344: 1676–1679. PubMed Abstract \| Publisher Full Text\n\nFurlan JC, Branddo LG, Ferraz AR, et al.: Surgical anatomy of the extralaryngeal aspect of the superior laryngeal nerve. Arch. Otolaryngol. Head Neck Surg. 2003; 129: 79–82. PubMed Abstract \| Publisher Full Text\n\nKiray A, Naderi S, Ergur I, et al.: Surgical anatomy of the internal branch of superior laryngeal nerve. Eur. Spine J. 2006; 15: 1320–1325. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun W, Wen W-P, Zhu X-L: Preservation of Internal Branch of Superior Laryngeal Nerve during Surgery for Hypopharyngeal Cancer. Ear, Nose & Throat Journal. 2022; 101(3): 175–180.\n\nParaskevas GK, Raikos A, Ioannidis O, et al.: Topographic anatomy of the internal laryngeal nerve: surgical considerations. Head Neck. 2012; 34(4): 534–540. PubMed Abstract \| Publisher Full Text\n\nGuven EM, Karacan K, Guven M, et al.: Topographic anatomy of the internal branch of superior laryngeal nerve. Eur Arch Otorhinolaryngeol. 2021; 278(3): 727–731. PubMed Abstract \| Publisher Full Text\n\nStephens RE, Wendel KH, Addington WR: Anatomy of the internal branch of the superior laryngeal nerve. Clin. Anat. 1999; 12: 79–83. Publisher Full Text\n\nPotenza AS, Araujo Filho VJ, Cernea CR: Injury of the external branch of the superior laryngeal nerve in thyroid surgery. Gland Surg. 2017; 6(5): 552–562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomassin JM: Le nerf larynge superieur. J. Fr. Otorhinolaryngol. Audiophonol. Chir. Maxillofac. 1982; 31: 139–150.\n\nKambic V, Zargi M, Radse IZ: Topographic anatomy of the external branch of the superior laryngeal nerve: its importance in head and neck surgery. J. Laryngol. Otol. 1984; 98: 1121–1124. PubMed Abstract \| Publisher Full Text\n\nCernea CR, Nishio S, Hojaij FG: Identification of the EBSLN in large goiters. Am. J. Otol. 1995; 16: 307–311. PubMed Abstract \| Publisher Full Text\n\nKierner AC, Aiger M, Burian M: The EBSLN: its topographical anatomy as related to surgery of the neck. Arch. Otolaryngol. Head Neck Surg. 1998; 124: 301–303. Publisher Full Text\n\nCha YH, Moon SY, Jehoon O, et al.: Anatomy of the external branch of the superior laryngeal nerve in Asian population. Sci. Rep. 2017; 7(1): 14952. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedman M, Lo Savio P, Ibrahim H: Superior laryngeal nerve identification and preservation in thyroidectomy. Arch. Otolaryngol. Head Neck Surg. 2002; 128(3): 296–303. Publisher Full Text\n\nJonas J, Bahr R: Neuromonitoring of the EBSLN during thyroid surgery. Am. J. Surg. 2000; 179: 234–236. Publisher Full Text\n\nLore JM, Kokocharov SI, Kaufman S, et al.: 38-Year evaluation of a surgical technique to protect the EBSLN during thyroidectomy. Ann. Otol. Rhinol. Laryngol. 1998; 107: 1015–1022. PubMed Abstract \| Publisher Full Text\n\nJiang Y, Gao B, Zhang X, et al.: Prevention and treatment of recurrent laryngeal nerve injury in thyroid surgery. Int. J. Clin. Exp. Med. 2014; 7(1): 101–107. PubMed Abstract\n\nCulp JM, Patel G: Recurrent Laryngeal Nerve Injury. [Updated 2023 May 22]. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. Reference Source\n\nJoliat GR, Guarnero V, Demartines N, et al.: Recurrent laryngeal nerve injury after thyroid and parathyroid surgery: Incidence and postoperative evolution assessment. Medicine (Baltimore). 2017; 96(17): e6674. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenninger MS, Gillen JB, Altman JS: Changing etiology of vocal fold immobility. Laryngoscope. 1998; 108(9): 1346–1350. PubMed Abstract \| Publisher Full Text\n\nNorris BK, Schweinfurth JM: Arytenoid dislocation: An analysis of the contemporary literature. Laryngoscope. 2011; 121(1): 142–146. PubMed Abstract \| Publisher Full Text\n\nSailaja K: A study on internal laryngeal nerve-its variation in the course, branching, anastamosis and relation to inferior thyroid artery. Int J Res Med Sci. 2016; 4: 4602–4606.\n\nShao T, Qiu W, Yang W: Anatomic variants of the recurrent laryngeal nerves in Chinese patients: a prospective study of 2,404 patients. Sci. Rep. 2016; 6: 25475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsgharpour E, Maranillo E, Sañudo J, et al.: Recurrent laryngeal nerve landmarks revisited. Head Neck. 2012; 34: 1240–1246. PubMed Abstract \| Publisher Full Text\n\nTitche LL: Causes of recurrent laryngeal nerve paralysis. Arch. Otolaryngol. 1976; 102: 259–261. Publisher Full Text\n\nFriedrich T, Steinert M, Keitel R, et al.: Incidence of damage to the recurrent laryngeal nerve in surgical therapy of various thyroid gland diseases—a retrospective study. Zentralbl. Chir. 1998; 123(1): 25–29. PubMed Abstract\n\nZakaria HM, Al Awad NA, Al Kreedes AS, et al.: Recurrent laryngeal nerve injury in thyroid surgery. Oman Med. J. 2011; 26(1): 34–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampos BA, Henriques PR: Relationship between the recurrent laryngeal nerve and the inferior thyroid artery: a study in corpses. Rev. Hosp. Clin. Fac. Med. Sao Paulo. 2000; 55(6): 195–200. Publisher Full Text\n\nSkandalakis JE, Droulias C, Harlaftis N, et al.: The recurrent laryngeal nerve. Am. Surg. 1976; 42(9): 629–634. PubMed Abstract\n\nSteurer M, Passler C, Denk DM, et al.: Advantages of recurrent laryngeal nerve identification in thyroidectomy and parathyroidectomy and the importance of preoperative and postoperative laryngoscopic examination in more than 1000 nerves at risk. Laryngoscope. 2002; 112(1): 124–133. PubMed Abstract \| Publisher Full Text\n\nÇakir BO, Ercan I, Şam B, et al.: Reliable surgical landmarks for the identification of the recurrent laryngeal nerve. Otolaryngol. Head Neck Surg. 2006; 135: 299–302. PubMed Abstract \| Publisher Full Text\n\nHenry BM, Vikse J, Graves JM, et al.: Extralaryngeal branching of the recurrent laryngeal nerve: a meta-analysis of 28,387 nerves. Langenbeck's Arch. Surg. 2016; 401: 913–923. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRai R: Morphometry of laryngeal nerves in cadavers. Dataset. figshare. 2024. Publisher Full Text" }	[ { "id": "301392", "date": "18 Sep 2024", "name": "Özcan Gayretli", "expertise": [ "Reviewer Expertise Anatomy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, I congratulate the work team because they produce a work that requires time and effort. The region studied is an area where surgical complications are common. In this respect, even small contributions to the regional anatomy can lead to important results. Although the measurement parameters will not contribute significantly to the literature, the variations and described morphological parameters are valuable.\nI would like to thank you for providing me with the opportunity to evaluate this study. King regards.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "12470", "date": "19 Sep 2024", "name": "Dr. Rajalakshmi Rai", "role": "Author Response", "response": "Dear Sir, Thank you for taking your valuable time to review the article and approving it. Your positive comments are inspiring and very much appreciated. With warm regards, Dr. Rajalakshmi Rai" } ] } ]	1	https://f1000research.com/articles/13-770
https://f1000research.com/articles/13-769/v1	08 Jul 24	{ "type": "Research Article", "title": "Comparative hemodynamics of ATTR and AL amyloidosis with exercise-induced pulmonary hypertension: A retrospective analysis", "authors": [ "Arif Albulushi", "Amna Al-Busaidi", "Kumayl Al-Lawatia", "Thuraiya Al-Rawahi", "Matlooba Al-Zadjali", "Amna Al-Busaidi", "Kumayl Al-Lawatia", "Thuraiya Al-Rawahi", "Matlooba Al-Zadjali" ], "abstract": "Background Cardiac amyloidosis, characterized by the deposition of amyloid proteins in the heart tissue, presents in two main types: transthyretin (ATTR) and light-chain (AL) amyloidosis. The hemodynamic response to exercise and the relationship with pulmonary hypertension (PH) in these patients is not well understood.\n\nMethods This retrospective study analyzed 100 patients diagnosed with either ATTR or AL amyloidosis. We assessed the prevalence of PH at rest and its induction during exercise stress tests. Hemodynamic parameters were measured to identify differences in the cardiac response to exercise between the two subtypes.\n\nResults A higher prevalence of PH was noted in the ATTR group compared to the AL group. Exercise stress tests induced significant PH, particularly in the ATTR subgroup. Subtle yet clinically relevant hemodynamic differences were observed between the amyloidosis subtypes.\n\nConclusions Our findings suggest that the amyloidosis subtype is an important factor in the management of PH. There is a need for tailored clinical approaches to address the distinct pathophysiological mechanisms in ATTR and AL amyloidosis. This study contributes to a better understanding of the hemodynamic changes during exercise in cardiac amyloidosis and underscores the importance of subtype-specific management strategies.", "keywords": [ "Cardiac Amyloidosis", "Chronic Heart Failure", "Exercise-Induced PH", "Hemodynamic Response" ], "content": "Introduction\n\nCardiac amyloidosis, typified by ATTR (transthyretin) and AL (light-chain) amyloidosis, represents a spectrum of conditions characterized by extracellular deposition of misfolded proteins in the cardiac matrix, leading to diastolic dysfunction and restrictive cardiomyopathy.1,2 The intricacies of cardiac amyloidosis, coupled with its clinical manifestations, present a diagnostic challenge, often leading to delayed or missed diagnoses.3\n\nRecent research underscores the prevalence of pulmonary hypertension (PH) in patients with cardiac amyloidosis, revealing its potential impact on prognosis and management strategies.4 ATTR amyloidosis, a hereditary or wild-type form, and AL amyloidosis, associated with plasma cell dyscrasia, each have distinct pathophysiological profiles but share the common complication of PH, contributing to increased morbidity and mortality.5\n\nThe hemodynamic burden imposed by amyloid deposition in the myocardium is not fully understood, especially under conditions of stress or exercise.6 This study aims to elucidate the hemodynamic changes associated with ATTR and AL amyloidosis at rest and during exercise, examining the prevalence of PH and its induction through stress testing to better understand the physiological demands placed on the heart in the presence of amyloid fibril infiltration.\n\nBy exploring the interplay between ATTR and AL amyloidosis with exercise-induced PH, this study aims to provide deeper insights into the hemodynamic alterations and their clinical implications, thereby informing more targeted therapeutic approaches for this patient population.\n\n\nMethods\n\nThis retrospective cohort study analyzes the hemodynamic data from ATTR and AL amyloidosis patients between January 2015 and December 2023. Our primary aim was to compare the prevalence and severity of pulmonary hypertension (PH) and exercise-induced hemodynamic changes between the two amyloidosis types.\n\n100 patients diagnosed with cardiac amyloidosis, confirmed via biopsy, were included. Of these, 40 had ATTR amyloidosis and 60 had AL amyloidosis.\n\nAll patients underwent right heart catheterization (RHC) at rest. A subset of 70 patients also participated in exercise tests to induce PH. Hemodynamic parameters including mean pulmonary artery pressure (mPAP), pulmonary capillary wedge pressure (PCWP), and right atrial pressure (RAP) were recorded. The change in mPAP relative to cardiac output (ΔmPAP/ΔCO) was used to assess the exercise-induced PH. Exercise-induced pulmonary hypertension (PH) is characterized by an increase in mean pulmonary arterial pressure (mPAP) to >30 mmHg at a cardiac output (CO) of <10 L/min during exercise, or a total pulmonary vascular resistance (TPVR) >3 Wood units (WU). Additionally, a rise in mean pulmonary arterial pressure relative to the increase in cardiac output (ΔmPAP/ΔCO) exceeding 3 mmHg/L/min, and an elevation in pulmonary arterial wedge pressure (ΔPAWP/ΔCO) surpassing 2 mmHg/L/min from rest to exercise, indicate post-capillary exercise-induced PH.7\n\nDifferences in PH prevalence between ATTR and AL groups were compared using the chi-square test, and exercise-induced hemodynamic changes were analyzed with the Mann-Whitney U test. A p-value of less than 0.05 was considered statistically significant.\n\nThis study was approved by IRB at UNMC, Ethical Approval Committee: 0839-21-CB (12/2/2021). Due to the retrospective nature of the data analysis, the requirement for informed consent was waived. The study adhered to the ethical principles outlined in the Declaration of Helsinki for medical research involving human subjects.\n\n\nResults\n\nOur study's cohort featured 100 patients with cardiac amyloidosis, 40 with transthyretin amyloidosis (ATTR), and 60 with light-chain amyloidosis (AL) otherwise no difference in demographics Table 1. The incidence of pulmonary hypertension (PH) was higher in the ATTR group at 66% compared to 57% in the AL group, a difference that was on the cusp of statistical significance (p=0.051) Figure 1.\n\nUpon exercise stress testing, PH was elicited in the entirety of the ATTR subgroup and 90% of the AL subgroup, marking a significant differential response to physical exertion (p=0.04). Detailed hemodynamic profiling during exercise unveiled a nuanced interplay between ventricular response and vascular load. The median increase in mean pulmonary arterial pressure relative to cardiac output (ΔmPAP/ΔCO) was marginally higher for ATTR patients. Similarly, the median rise in pulmonary artery wedge pressure per unit cardiac output (ΔPAWP/ΔCO) also favored ATTR amyloidosis, though this did not reach a level of statistical significance Figure 2.\n\nNotably, the median increase in right atrial pressure per unit cardiac output (ΔRAP/ΔCO) was marginally higher in the AL group, reflecting a potential difference in central venous pressure dynamics during stress between the amyloidosis types. Nonetheless, the ratio of ΔRAP to ΔPAWP remained consistent across both groups, suggesting a uniform relationship between right atrial pressures and left-sided filling pressures during exercise, regardless of amyloid type (p=0.08) Table 2.\n\nThese findings illuminate the pathophysiological divergence in exercise response between ATTR and AL amyloidosis, underscoring the need for tailored clinical management strategies.\n\n\nDiscussion\n\nThis study's findings reveal a complex interplay between cardiac amyloidosis subtypes and pulmonary hypertension (PH), particularly under exercise conditions.8 The higher prevalence of exercise-induced PH in ATTR compared to AL amyloidosis may reflect the distinct pathophysiological mechanisms at play.9 ATTR amyloidosis, with its non-mutant and mutant variants, often leads to stiffer ventricular walls, predisposing patients to a higher incidence of PH during stress.7\n\nThe marginal differences in hemodynamic responses, such as ΔmPAP/ΔCO and ΔPAWP/ΔCO, suggest a possible disparity in how each amyloid subtype affects cardiac and vascular function.10 These disparities could be attributed to the differential myocardial infiltration patterns and the resultant impact on ventricular compliance and contractility. The similar ΔRAP/ΔPAWP ratios between the groups might indicate a shared mechanism in the way increased left heart pressures are transmitted back to the right heart, despite the differences in amyloid fibril composition anetd deposition.\n\nThe borderline statistical significance observed in PH prevalence and hemodynamic responses warrants further investigation. A larger sample size could elucidate whether the trends observed are consistent and statistically robust across a broader population.\n\nClinically, these findings emphasize the importance of personalized management strategies for PH in patients with cardiac amyloidosis.4 They also raise questions about the potential benefits of targeted therapies for PH that consider the specific amyloidosis subtype. Prospective studies might investigate whether treatments that are effective for PH in one subtype may be less so in another, due to the underlying molecular and structural differences.\n\nThis research contributes to a growing body of evidence that suggests cardiac amyloidosis, particularly ATTR, is an underrecognized etiology in patients presenting with PH.10 Recognition of this relationship is critical, as it may guide the timing of diagnostic procedures like RHC, especially when noninvasive assessments are inconclusive or suggest PH. It also underscores the need for heightened clinical awareness of amyloidosis as a differential diagnosis in PH patients.\n\n\nConclusion\n\nThe hemodynamic response to exercise in cardiac amyloidosis patients with PH presents a multifaceted challenge. Our study adds to the understanding of this response, providing a foundation for future research and clinical practice that could improve outcomes for this patient population.\n\n0839-21-CB (12/2/2021).\n\nAuthors AA & KS were involved in the conception and design, and author HA was involved in the analysis and interpretation of the data; the drafting of the paper, revising it critically for intellectual content; and the final approval of the version to be published. All authors agree to be accountable for all aspects of the work.\n\nThis study was approved by IRB at UNMC, Ethical Approval Committee: 0839-21-CB (12/2/2021). Due to the retrospective nature of the data analysis, the requirement for informed consent was waived. The study adhered to the ethical principles outlined in the Declaration of Helsinki for medical research involving human subjects.\n\nOur study involves data that are sensitive in nature due to [patient confidentiality, proprietary information]. To protect the privacy and rights of the individuals involved and to comply with [local institute regulations and guidelines], access to the complete dataset is restricted.\n\nResearchers or reviewers interested in accessing the dataset must:\n\n1. Submit a formal request to [specify the authority, e.g., the principal investigator, a specific department, or an institutional review board] outlining the purpose of the data request.\n\n2. Provide a detailed research proposal, including objectives, methodology, and expected outcomes.\n\n3. Agree to abide by all ethical guidelines, including [specify any specific requirements, such as data handling procedures, confidentiality agreements, etc.].\n\n4. Obtain approval from their own institutional review board (IRB) or ethics committee, if applicable.\n\nAccess to the data will be granted under the following conditions:\n\n1. The data will be used solely for the purposes outlined in the approved research proposal.\n\n2. Any publication or dissemination of results derived from the data must acknowledge the source of the data.\n\n3. The data must not be shared with third parties or used to identify individual participants.\n\n4. All researchers accessing the data agree to comply with [specify any additional conditions, such as data security measures, reporting requirements, etc.].\n\nWe believe these measures are necessary to ensure the ethical use of the data and to protect the privacy and security of the individuals and entities involved. Should you require any further information or clarification, please do not hesitate to contact us.", "appendix": "Data availability statement\n\nData available upon request by contacting the correspondence author via dr.albulushi@gmail.com. Due to ethical restrictions and the sensitive nature of the clinical data, these are not publicly deposited but are available under conditions that preserve the privacy of individuals involved.\n\n\nReferences\n\nMeaney E, Shabetai R, Bhargava V, et al.: Cardiac amyloidosis, constrictive pericarditis, and restrictive cardiomyopathy. Am. J. Cardiol. 1976; 38(5): 547–556. Publisher Full Text\n\nAkatsuka T, Fujimoto N, Ishiyama M, et al.: Trajectory of left ventricular geometry and diastolic dysfunction in hereditary transthyretin cardiac amyloidosis. ESC Heart Fail. 2021; 8(4): 3422–3426. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPorcari A, et al.: Cardiac amyloidosis: do not forget to look for it. Eur. Heart J. Suppl. 2020; 22(Suppl_E): E142–E147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDingli D, Utz JP, Gertz MA: Pulmonary hypertension in patients with amyloidosis. Chest. 2001; 120(5): 1735–1738. Publisher Full Text\n\nSlivnick J, et al.: Prevalence and haemodynamic profiles of pulmonary hypertension in cardiac amyloidosis. Open Heart. 2022; 9(1): e001808. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanydeen R, et al.: Diagnostic and prognostic values of cardiopulmonary exercise testing in cardiac amyloidosis. Front Cardiovasc Med. 2022; 9: 898033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolt MF, Flø A, Ravnestad H, et al.: Invasive haemodynamics at rest and exercise in cardiac amyloidosis. ESC Heart Fail. 2023 Dec 29; 11: 1263–1268. Publisher Full Text\n\nCantone A, Serenelli M, Sanguettoli F, et al.: Cardiopulmonary exercise testing predicts prognosis in amyloid cardiomyopathy: a systematic review and meta-analysis. ESC Heart Fail. 2023; 10(4): 2740–2744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBustamante JG, Zaidi SRH: Amyloidosis. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 Jul 31.\n\nLonginow J, et al.: Significance of Pulmonary Hypertension in Cardiac Amyloidosis. Am. J. Cardiol. 2023; 192: 147–154. PubMed Abstract \| Publisher Full Text" }	[ { "id": "314999", "date": "04 Sep 2024", "name": "Selda Murat", "expertise": [ "Reviewer Expertise cardiomyopathy" ], "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 methodology of the study is not suitable for retrospective data recording studies. It should be explained for which indications right catheter and exercise-induced catheter were performed after diagnosis.\nIf these interventional procedures were performed within the scope of this study, the study design should be stated prospectively.\nThe study ethics committee date is written as 2021, but the data collecting intervals in which the study was performed are stated as 2015-2023. This is confusing.\nThe discussion is very insufficient.\nIt would be appropriate to provide information about the patients' right heart functions (TAPSE, RV area, RV strain ..).\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?\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? No", "responses": [ { "c_id": "12373", "date": "05 Sep 2024", "name": "SS DM2020", "role": "Author Response", "response": "1. Methodology of the study is not suitable for retrospective data recording studies. Response: Thank you for pointing this out. We recognize that the retrospective nature of our study may have caused some confusion in the presentation. The study involved a review of existing patient data where right heart catheterization (RHC) and exercise-induced RHC were performed as part of routine clinical care after diagnosing cardiac amyloidosis. We will update the methodology section to clarify that these procedures were not part of a prospective study but rather data were gathered retrospectively from patients who had undergone RHC as part of their diagnostic workup. This will ensure the distinction between retrospective data collection and prospective study designs is clear. 2. It should be explained for which indications right heart catheterization and exercise-induced catheterization were performed after diagnosis. Response: We appreciate your comment and agree that this needs to be clarified. We will add a detailed explanation regarding the indications for performing RHC and exercise-induced catheterization. Typically, these procedures were performed in patients with suspected or established pulmonary hypertension (PH) based on non-invasive imaging findings, worsening symptoms, or unexplained dyspnea, especially during exertion. We will include this information in the revised manuscript to better explain the rationale behind the selection of patients for these procedures. 3. If these interventional procedures were performed within the scope of this study, the study design should be stated prospectively. Response: We acknowledge the confusion caused by the presentation of the study design. As noted earlier, this was a retrospective study, and no interventional procedures were conducted specifically for the purpose of the study. The data were gathered from patients who had already undergone RHC and exercise-induced catheterization as part of their clinical care. We will explicitly state this in the study design section to avoid any ambiguity. 4. The study ethics committee date is written as 2021, but the data collection intervals in which the study was performed are stated as 2015-2023. This is confusing. Response: You are correct in identifying this inconsistency. We will revise the manuscript to explain that the data collection was retrospective, spanning from 2015 to 2023, and that ethical approval was obtained in 2021 for the retrospective review of these data. This clarification will make the timeline more transparent. Thank you for sharing the reviewer's feedback. Below is a structured response to each of the concerns raised, which you can use as a guide to address the reviewer's comments effectively. Reviewer Concerns and Suggested Responses 1. Methodology of the study is not suitable for retrospective data recording studies. Response: Thank you for pointing this out. We recognize that the retrospective nature of our study may have caused some confusion in the presentation. The study involved a review of existing patient data where right heart catheterization (RHC) and exercise-induced RHC were performed as part of routine clinical care after diagnosing cardiac amyloidosis. We will update the methodology section to clarify that these procedures were not part of a prospective study but rather data were gathered retrospectively from patients who had undergone RHC as part of their diagnostic workup. This will ensure the distinction between retrospective data collection and prospective study designs is clear. 2. It should be explained for which indications right heart catheterization and exercise-induced catheterization were performed after diagnosis. Response: We appreciate your comment and agree that this needs to be clarified. We will add a detailed explanation regarding the indications for performing RHC and exercise-induced catheterization. Typically, these procedures were performed in patients with suspected or established pulmonary hypertension (PH) based on non-invasive imaging findings, worsening symptoms, or unexplained dyspnea, especially during exertion. We will include this information in the revised manuscript to better explain the rationale behind the selection of patients for these procedures. 3. If these interventional procedures were performed within the scope of this study, the study design should be stated prospectively. Response: We acknowledge the confusion caused by the presentation of the study design. As noted earlier, this was a retrospective study, and no interventional procedures were conducted specifically for the purpose of the study. The data were gathered from patients who had already undergone RHC and exercise-induced catheterization as part of their clinical care. We will explicitly state this in the study design section to avoid any ambiguity. 4. The study ethics committee date is written as 2021, but the data collection intervals in which the study was performed are stated as 2015-2023. This is confusing. Response: You are correct in identifying this inconsistency. We will revise the manuscript to explain that the data collection was retrospective, spanning from 2015 to 2023, and that ethical approval was obtained in 2021 for the retrospective review of these data. This clarification will make the timeline more transparent. 5. The discussion is very insufficient. Response: We appreciate your feedback on the discussion. To address this, we will expand the discussion to include: A deeper analysis of the differences in hemodynamic responses between ATTR and AL amyloidosis during exercise. A comparison with other studies on cardiac amyloidosis and pulmonary hypertension to put our findings into context. Clinical implications of our results, particularly how they might inform tailored management strategies for ATTR and AL amyloidosis patients. This revision will aim to provide a more comprehensive interpretation of the findings and their significance in current clinical practice. 6. It would be appropriate to provide information about the patients' right heart functions (TAPSE, RV area, RV strain). Response: Thank you for this important suggestion. We agree that including parameters of right heart function such as tricuspid annular plane systolic excursion (TAPSE), right ventricular (RV) area, and RV strain would add valuable insight to the study. Although this study primarily focused on \"invasive\" pulmonary hemodynamics, we will re-examine the dataset to extract information on these parameters. If available, we will present these data in the results section and discuss the relevance of right heart function in the context of exercise-induced PH and amyloidosis. 7. Is the work clearly and accurately presented and does it cite the current literature? Response: We acknowledge that the manuscript may not have fully addressed the current literature. In the revised version, we will ensure that recent and relevant studies on cardiac amyloidosis, PH, and exercise-induced hemodynamics are cited. This will provide a stronger foundation for our discussion and interpretation of the results. 8. Are the conclusions drawn adequately supported by the results? Response: We recognize that the conclusions may not be fully supported by the current results due to the limitations of sample size and some borderline statistical findings. In the revision, we will adjust our conclusions to more cautiously reflect the data, emphasizing trends and the need for further research to confirm our findings. We will also include a clearer discussion of the study’s limitations." } ] } ]	1	https://f1000research.com/articles/13-769
https://f1000research.com/articles/13-522/v1	22 May 24	{ "type": "Study Protocol", "title": "Identifying the role of Phytomolecules in the management of liver diseases by modulating NRF2 pathway: A Scoping Review Protocol", "authors": [ "Ajay Mili", "Priyobrat Rajkhowa", "Krishnadas Nandakumar", "Richard Lobo", "Ajay Mili", "Priyobrat Rajkhowa", "Krishnadas Nandakumar" ], "abstract": "Background The Liver is a vital organ in the human body, which plays a crucial role in various physiological processes. Oxidative stress is a critical factor in the pathogenesis and progression of various liver diseases, contributing to cellular damage and dysfunction. The Liver is particularly vulnerable to the harmful effects of reactive oxygen species when the balance between their production and the body’s antioxidant defense mechanisms is disrupted. The nuclear factor erythroid 2-related factor 2 (NRF2) pathway has emerged as a promising therapeutic target for liver diseases due to its pivotal role in cellular defense against oxidative stress and inflammation. Plants have always been a source of drugs which has been used to treat various pharmacological disorders and most of its activity is due to its potential as an antioxidant. However, the specific mechanisms by which they interact with the NRF2 pathway and confer protection against liver diseases remain inadequately elucidated. Therefore, this scoping review aims to identify and analyze the existing literature pertaining to the relationship between Phytomolecules, which can modulate NRF2 and protect against liver diseases.\n\nMethods The proposed scoping review will follow the steps given by “Arksey and O’Malley and Levac et al”. Electronic databases (PubMed/MEDLINE, Embase, etc.) will be searched for recent relevant studies. A predefined criterion for the inclusion and exclusion of studies will be independently adopted by two reviewers. The review will be presented as per the “Preferred Reporting Items for Systematic Reviews and Meta-analyses Extension for Scoping Review (PRISMA-ScR)” guidelines.\n\nConclusion The scoping review finding is expected to help understanding the role of Phytomolecules in preventing liver diseases by modulating the NRF2 pathway. Ultimately, this review will serve as a foundational step toward developing targeted interventions to improve liver health outcomes and reduce the global burden of liver diseases.", "keywords": [ "NRF2 pathway", "Liver diseases", "Phytomolecules", "Oxidative stress" ], "content": "Introduction\n\nLiver diseases represent a significant global health challenge, including a variety diseases including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), drug-induced liver injury (DILI), and liver cirrhosis.1,2 The liver, a pivotal metabolic organ, plays an important role in physiological processes including detoxification, metabolic regulation, protein biosynthesis, bile secretion, and immunomodulation. Intracellular reactive oxygen species (ROS) are produced internally, for instance during mitochondrial oxidative phosphorylation, or can emerge through interactions with external agents such as xenobiotic substances. For maintain the ROS levels, Liver has various antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase (SOD). Any imbalance in the working of the antioxidant enzymes can lead to oxidative stress and it is one of the key factors in the progression and onset of liver diseases.3,4\n\nThe Nuclear factor erythroid 2-related factor 2 (NRF2) pathway has emerged as a promising target for managing liver diseases. NRF2, a transcription factor, is integral to cellular defense mechanisms against oxidative stress and inflammation. Upon activation, NRF2 facilitates the expression of various genes involved in antioxidant and detoxification processes, thereby increasing the synthesis of antioxidant enzymes and proteins that mitigate the negative impacts of ROS and pro-inflammatory mediators. Growing evidence implicates NRF2 pathway dysregulation in liver disease pathogenesis and progression. Both experimental and clinical investigations have explored NRF2 modulation to mitigate liver damage, attenuate inflammation, and enhance hepatocyte viability.5,6 Consequently, identifying compounds capable of activating the NRF2 pathway holds potential for advancing liver disease management strategies.\n\nPhytomolecules, also known as phytochemicals or secondary metabolites, have recently been gaining a distinguished status as a potential source of drugs for treating various diseases. Phytomolecules have various pharmacological activities, such as antioxidant,7,8 anti-inflammatory,7 anticancer,9,10 Cardioprotective,11,12 Neuroprotective,13,14 Hepatoprotective,15,16 through their potential as an antioxidant and the potential to alter the signaling pathways.\n\nIt is essential to note that the precise role of Phytomolecules as modulators of the NRF2 pathway in managing liver diseases remains uncertain or ambiguous. Consequently, this scoping review is being undertaken to address this knowledge gap. By amalgamating existing literature on the role of Phytomolecules as modulators of the NRF2 pathway in managing liver diseases through the adoption of scoping review methodology, this study distinguishes itself and contributes novel evidence to the field.\n\n\nProtocol\n\nThe protocol for the scoping review has been formulated by utilizing the frameworks proposed by “Arksey and O’Malley and Levac et al.”.17 This review will adhere to the guidelines outlined in the Preferred Reporting Item for Scoping Reviews (PRISMA-ScR). For ensuring proper reporting, the “Preferred Reporting Items for Scoping Reviews (PRISMA-ScR)” will be adopted.18\n\nThe review question was framed through systematic brainstorming and iteratively refining ideas by the team. A literature search on Phytomolecules, NRF2 and Liver disease was done, leading to our research question.\n\n1. What are the different types of Phytomolecules that contribute to the modulation of the NRF2 pathway for protection against Liver disease?\n\nJBI manual for evidence synthesis 2020 format will be adopted for the development of research question PCC (Population, Concept, Context) (Table 1).19 The study’s primary goal is to methodically identify and map the existing literature on the role of Phytomolecules in managing liver diseases through the modulation of the NRF2 pathway.\n\nA literature search will be done in the electronic database, including Scopus, Web of science, CINAHL, PubMed, and EMBASE, to identify recent relevant literature published from 2015 to 2024. The keywords which will be used for searching are listed in Table 2. The search will focus exclusively on articles written in English. We’ll conduct a comprehensive exploration of potentially relevant articles by scrutinizing the reference lists of the included papers. Rayyan, an online platform, will be utilized to manage the collected data.20\n\n* Wildcard character of Boolean search to include alternative form of word.\n\nScoping reviews are broad by nature, so searching, screening, and selecting studies can uncover new terms, ideas, and even sources of information that weren’t initially considered. The search strategy can be modified throughout the review process to keep up with these findings.\n\nAfter the initial literature search, all retrieved citations will be aggregated and uploaded to the Rayyan platform that will help in efficient literature management, including duplicate removal, study screening and selection, and collaborative review among researchers.\n\nTwo reviewers, AM & PR will screen the title and abstract of the retrieved papers using a pre-defined inclusion and exclusion criteria in the Rayyan platform. In case of conflict regarding an article, an independent or 3rd reader will be sought. The entirety of eligible research documents will be acquired subsequent to the preliminary assessment and will be subjected to independent evaluation by two reviewers. Included studies will undergo data charting and be reported by adhering to the PRISMA 2020 diagram.21 All studies meeting the inclusion and exclusion criteria, regardless of quality, will be incorporated (Table 3).\n\nThe process of data charting will be executed utilizing a predetermined format designed for this purpose. Two reviewers will independently chart data for each paper included in the review using Microsoft excel. In the case of any disagreement, the opinion of an additional or the third reviewer will be sought. This systematic procedure will assist in identifying gaps in the research area. The data extraction will include i) Study title, ii) Authors, iii) Publication year, iv) Study design, v) Study objectives, vi) Findings, vii) Study subjects, viii) Liver diseases studied, ix) Type of phytomolecules, x) Phytomolecule source, xi) NRF2 pathway modulation, xii) Outcome measures, xiii) Conclusion, and xiv) Recommendations.\n\nThe data extraction protocol will be iteratively refined as data is obtained from each included source of evidence. Any adjustments made will be meticulously documented within the scoping review. As a pragmatic measure, there will be no direct outreach to included study authors to solicit missing or supplementary data.\n\nThe study findings will be synthesized using a combination of narrative and tabular formats to present the generated evidence. Qualitative and quantitative evidence from the developed data charting table will help in mapping the accessible literature and reporting studies that depict the key findings pertaining to the roles of Phytomolecules as NRF2 modulator in the management of liver diseases. The process will involve identifying knowledge gaps or areas necessitating further research. Additionally, consider disseminating the findings through publication in a peer-reviewed journal or presentation at relevant conferences.\n\n\nDiscussion\n\nThroughout history, humans have utilized plants as a source of beneficial molecules, including Phytomolecules. Therefore, the present scoping review seeks to identify and document the Phytomolecules that can protect against liver disease by modulating the NRF2 pathway. The review’s findings will help provide a comprehensive overview of the Plant, its potential Phytomolecules, class of Phytomolecules, and the type of liver disease that can be treated or managed by Phytomolecules by NRF2 modulation. By providing early insights into a drug’s potential and guiding its development, they significantly increase the chances of success while ensuring ethical considerations and regulatory compliance, paving the way for developing safe and effective treatments.\n\nThe limitations of this review are that it excludes non-English databases in the literature search as it can cause practical implications, and neither will assess the quality of studies methodology.\n\nFindings will be disseminated in a scientific journal.\n\nThe literature search is ongoing.\n\nAs there are no human participants involved, there will be no requirement for ethical approval. Patients and/or the public were not involved in the design of this protocol.\n\n\nAuthors contribution\n\nAjay Mili: Conceptualization, Methodology, Investigation, Data Curation, Writing- Original draft preparation, Writing-Reviewing and Editing. Priyobrat Rajkhowa: Methodology, Investigation, Data Curation, Writing-Reviewing and Editing. Krishnadas Nandakumar: Supervision, Data Curation, Writing-Reviewing and Editing. Richard Lobo: Supervision, Data Curation, Writing-Reviewing and Editing.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nOpen Science Framework: “PRISMA-ScR-Fillable-Checklist_10Sept2019”, https://doi.org/10.17605/OSF.IO/C3P8K.\n\n\nAcknowledgment\n\nAuthor Ajay Mili and Priyobrat Rajkhowa are thankful to the Manipal Academy of Higher Education (MAHE) for providing Dr.T.M.A. Pai Doctoral Fellowship. The authors also acknowledge with thanks the Manipal College of Pharmaceutical Sciences (MCOPS), Prasanna School of Public Health (PSPH), and the Manipal Academy of Higher Education (MAHE) for providing facilities for this work.\n\n\nReferences\n\nSharma A, Nagalli S: Chronic Liver Disease - StatPearls - NCBI Bookshelf. StatPearls; 2022.\n\nCheemerla S, Balakrishnan M: Global Epidemiology of Chronic Liver Disease. Clin. Liver Dis. 2021 May 4; 17(5): 365–370. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi S, Tan H-Y, Wang N, et al.: The Role of Oxidative Stress and Antioxidants in Liver Diseases. Int. J. Mol. Sci. 2015 Nov 2; 16(11): 26087–26124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllameh A, Niayesh-Mehr R, Aliarab A, et al.: Oxidative Stress in Liver Pathophysiology and Disease. Antioxidants. 2023 Aug 22; 12(9): 1653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu D, Xu M, Jeong S, et al.: The Role of Nrf2 in Liver Disease: Novel Molecular Mechanisms and Therapeutic Approaches. Front. Pharmacol. 2019 Jan 8; 9(January): 1–7. Publisher Full Text\n\nZhou J, Zheng Q, Chen Z: The Nrf2 Pathway in Liver Diseases. Front. Cell Dev. Biol. 2022 Feb 10; 10(February): 1–14. Publisher Full Text\n\nHuang L, Kim JH, You L, et al.: Anti-oxidative, anti-apoptotic, and anti-inflammatory activities of Connarus semidecandrus Jack ethanol extract in UVB-irradiated human keratinocytes. J. Ethnopharmacol. 2024 Mar 1; 321(August 2023): 117574. PubMed Abstract \| Publisher Full Text\n\nXu D-P, Li Y, Meng X, et al.: Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and Resources. Int. J. Mol. Sci. 2017 Jan 5; 18(1): 96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaeem A, Hu P, Yang M, et al.: Natural Products as Anticancer Agents: Current Status and Future Perspectives. Molecules. 2022 Nov 30; 27(23): 8367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou Y, Li Y, Zhou T, et al.: Dietary Natural Products for Prevention and Treatment of Liver Cancer. Nutrients. 2016 Mar 10; 8(3): 156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTavakoli R, Tabeshpour J, Asili J, et al.: Cardioprotective Effects of Natural Products via the Nrf2 Signaling Pathway. Curr. Vasc. Pharmacol. 2021 Sep; 19(5): 525–541. PubMed Abstract \| Publisher Full Text\n\nTomou E-M, Papakyriakopoulou P, Skaltsa H, et al.: Bio-Actives from Natural Products with Potential Cardioprotective Properties: Isolation, Identification, and Pharmacological Actions of Apigenin, Quercetin, and Silibinin. Molecules. 2023 Mar 5; 28(5): 2387. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDas S, Nahar L, Nath R, et al.: Neuroprotective natural products.Sarker SD; Nahar LBT-AR in MC, editors. Medicinal Natural Products: A Disease-Focused Approach. Academic Press; 2020; pp. 179–206. Publisher Full Text\n\nMarino A, Battaglini M, Moles N, et al.: Natural Antioxidant Compounds as Potential Pharmaceutical Tools against Neurodegenerative Diseases. ACS Omega. 2022 Aug 2; 7(30): 25974–25990. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandey B, Baral R, Kaundinnyayana A, et al.: Promising hepatoprotective agents from the natural sources: a study of scientific evidence. Egypt. Liver J. 2023 Mar 29; 13(1): 14. Publisher Full Text\n\nBachar SC, Bachar R, Jannat K, et al.: Hepatoprotective natural products.Sarker SD; Nahar LBT-AR in MC, editors. Medicinal Natural Products: A Disease-Focused Approach. Academic Press; 2020; pp. 207–249. Publisher Full Text\n\nLevac D, Colquhoun H, O’Brien KK: Scoping studies: advancing the methodology. Implement. Sci. 2010 Dec 20; 5(1): 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann. Intern. Med. 2018 Oct 2; 169(7): 467–473. PubMed Abstract \| Publisher Full Text\n\nPeters MDJ, Marnie C, Tricco AC, et al.: Updated methodological guidance for the conduct of scoping reviews. JBI Evid. Synth. 2020 Oct; 18(10): 2119–2126. Publisher Full Text\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan—a web and mobile app for systematic reviews. Syst. Rev. 2016 Dec 5; 5(1): 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29; 372. Publisher Full Text" }	[ { "id": "288724", "date": "14 Jun 2024", "name": "Tamer A. Addissouky", "expertise": [ "Reviewer Expertise biomedical" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPositive points: - The research question is clearly defined using the PCC framework which provides context and scope for the review. - An extensive search strategy is described using relevant databases and search terms to capture all available literature. - Clear inclusion/exclusion criteria are defined to filter relevant studies in a systematic manner. - A team-based approach is outlined for the different stages of title/abstract screening, full-text review, and data extraction to ensure objectivity. - Data will be charted using a standardized format and then summarised both narratively and quantitatively to synthesise evidence. - Findings will be disseminated through publication, adding value.\nNegative points:\n\n- Quality assessment of studies is not mentioned which is important to evaluate strength of evidence. - No mention of handling any disagreement between reviewers during study selection. - Only English literature will be included which can introduce language bias. - No plans to search grey literature sources which may contain some relevant data. - No qualitative analysis is proposed which could provide contextual insights beyond numbers.\nRecommendations: - Develop and apply a quality assessment tool to appraise rigour of primary studies. - Detail a clear process for resolving any disagreements between reviewers. - Consider translating the abstracts of relevant non-English studies to broaden scope. - Include searches of dissertations, conference abstracts and trials registries. - Use qualitative data analysis methods like thematic synthesis for narrative findings. - Engage stakeholders to obtain their perspectives and ensure findings are meaningful. - Pilot test all tools and processes on a sample of studies to address any issues early.\nIn summary, this protocol outlines a rigorous systematic approach. Enhancing the quality assessment process, broadening the scope and applying qualitative synthesis would strengthen the review.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11945", "date": "08 Jul 2024", "name": "Richard Lobo", "role": "Author Response", "response": "Dear Reviewer, Thank you for your detailed and constructive feedback on our scoping review protocol. We appreciate your insights and suggestions, which will undoubtedly strengthen the rigor and comprehensiveness of our study. Below are our responses and actions based on your specific points: Acknowledgment: We acknowledge your positive feedback on several aspects of our protocol: The research question is clearly defined using the PCC framework, providing a robust context and scope. An extensive search strategy is outlined, utilizing relevant databases and search terms to comprehensively capture available literature. Clear inclusion/exclusion criteria are defined to systematically filter relevant studies. A team-based approach is planned for screening, review stages, and data extraction to ensure objectivity. Data will be charted using a standardized format and synthesized both narratively and quantitatively. Findings will be disseminated through publication, adding value to the existing literature. Responses to Specific Questions: Quality Assessment of Studies: We acknowledge your concern regarding the quality assessment of individual studies in our scoping review. Please note that our review is focused on mapping the breadth of literature rather than conducting a formal quality assessment. The methodology follows the Arksey, H., & O’Malley, L. (2005) framework, which does not include the adoption of a quality assessment for studies. We aim to adhere to this established methodology. Furthermore, this scoping review serves as a preliminary step toward a systematic review. Upon completion of the scoping review, we plan to conduct a systematic review where we will incorporate a quality assessment tool to evaluate the included studies. Handling Disagreements Between Reviewers: We acknowledge your concern regarding the handling of disagreements between reviewers. This aspect is addressed in the study selection section of the methods section. We have now comprehensively detailed the process for handling disagreements between reviewers. Language Bias: We acknowledge your comment regarding the inclusion of studies published in various languages. We will attempt to include studies published in any language in the scoping review. However, the translation of these studies will rely on online translation tools. If we are unable to accurately translate certain articles, they will be excluded from the review. This limitation will be duly acknowledged in the limitations section of the manuscript. We have mentioned our approach to including any language of studies in the scoping review in the manuscript in Table 3. Grey Literature: We acknowledge the importance of including grey literature sources to capture additional relevant data. We have mentioned our approach to including grey literature in the manuscript in the search strategy section of the methods section. Qualitative Analysis: We appreciate your emphasis on the importance of qualitative analysis. While we initially proposed only narrative analysis, we recognize the value of incorporating additional qualitative data analysis methods, such as thematic synthesis, to provide deeper contextual insights. We have addressed this in the “Collating, summarizing, and reporting the results” section of the manuscript. Response to recommendations: Develop and apply a quality assessment tool to appraise the rigor of primary studies: We wish to clarify that our scoping review does not include a formal quality assessment of individual studies, as per the methodology outlined by Arksey and O’Malley (2005), which does not prescribe such an assessment. Implementing changes to this methodology at this stage poses challenges. Detail a clear process for resolving any disagreements between reviewers: We have embraced this recommendation and detailed a clear process for resolving disagreements between reviewers in the manuscript. Consider translating abstracts of relevant non-English studies to broaden scope: Regarding language bias, we have accepted the recommendation to consider translating abstracts of relevant non-English studies using online tools. If translation accuracy cannot be ensured, studies will be excluded as outlined in the manuscript. Include searches of dissertations, conference abstracts, and trials registries: We have accepted to include searches of grey literature sources such as dissertations, conference abstracts, and trial registries, as also specified in the manuscript. Consider qualitative data analysis methods like thematic synthesis: We have embraced the suggestion to incorporate qualitative data analysis methods like thematic synthesis, which has been highlighted in the manuscript. Engage stakeholders to ensure findings are meaningful: Upon completion of our study, we plan to consult various stakeholders to ensure that our findings are meaningful and impactful. Pilot test all tools and processes on a sample of studies to address any issues early: Recognizing its importance, we have conducted pilot testing on our tools, including the data extraction chart, to address any potential issues early in the process. We are committed to enhancing the transparency, rigor, and inclusivity of our scoping review methodology based on your valuable feedback. Please do not hesitate to reach out if you have any further questions or suggestions." } ] } ]	1	https://f1000research.com/articles/13-522
https://f1000research.com/articles/13-136/v1	22 Feb 24	{ "type": "Study Protocol", "title": "Study of molecular characterization for diagnosis of chronic and recurrent dermatophytosis", "authors": [ "Aditi Warghade", "Gargi Mudey" ], "abstract": "Dermatophytes are the keratinophilic fungi which infect humans and is the most recurring type of disease. The high level of transmissibility creates an epidemiological risk and emphasises the significance of these illnesses. However, a growing number of reports describing dermatophytes can cause deep infections in diabetic and immunocompromised patients, by invading deep layers like the dermis and hypodermis. Despite the prevalence and significance of dermatophytes in clinical mycology, it is not always possible to accurately diagnose this specific infection due to its overlapping structures among species of dermatophytes. Since it is difficult to identify species that exhibit weak characteristics in the morphological highlights, identification of the dermatophyte is often relied on its morphological analysis, which is a laborious process and demands skill. The massive shift in genetic variation, the source of infection, and epidemiological research can be discovered using molecular approaches. Therefore, the development of an accurate laboratory test for dermatophyte species identification is essential for the prevention and efficient management of dermatophytoses. One such methodology allows use of PCR technology which has many methods for molecular level characterization which is rapid, efficient, and capable of producing DNA polymorphisms specific to various dermatophyte species based on distinctive band patterns seen by agarose gel electrophoresis. The RAPD-PCR approach will be used in this study protocol to molecularly characterize the dermatophytes for precise speciation of the sample. In addition to improving knowledge of fungal biology and pathology with a focus on adaptive mechanisms to combat difficult conditions from host counteractions, there is a need to improve awareness of the importance of these diseases through accurate epidemiological data. The advantages of molecular approaches for characterizing objects over traditional methods are their sensitivity and specificity.", "keywords": [ "Dermatophytosis", "Molecular", "Characterisation", "RAPD-PCR", "Electrophoresis", "fungus", "keratinophilic", "Conventional" ], "content": "Introduction\n\nDifferent clinical symptoms caused by dermatophytes range from superficial to subcutaneous affliction, including tinea corporis, tinea capitis, tinea pedis, and tinea unguium.1 These diseases can range in severity clinically from moderate to severe depending on the host's immune system, the virulence of the strain, and other environmental factors.2–4 Although dermatophytoses harm people throughout, they are more common in tropical regions due to high temperatures and humidity.5 Age, sex, time of year, socioeconomic and cultural circumstances, and geographic location are all factors that might influence the development of dermatophytosis.6 Potassium hydroxide (KOH) direct microscopy followed by selective medium culture is the standard procedure for dermatophyte screening in clinical samples. A quick screening technique for fungal structures is microscopy performed directly on clinical specimens, but this method lacks specificity.7 By using phenotypic techniques, dermatophyte isolates can be classified according to genus or species based on colony characteristics, microscopic assessments, and biochemical tests like growth patterns.8 Because there are changes from one isolate to another and overlaps in the features of several species, dermatophyte species identification by morphology in cultures may be difficult or imprecise.9 Molecular-based methods rely on identifying genotypic variations in pathogenic organisms.10–12 Molecular methods are being used to identify dermatophytes since they are more precise than conventional techniques.13 The development of molecular diagnostics were encouraged by the conventional techniques for identifying dermatophytes which are imprecise and have a delayed diagnostic character. Techniques that permit for both the early and accurate detection of dermatophytosis in order to provide timely antifungal treatment that prevents generic over-the-counter medication.14 Therefore, it is essential to create more reliable dermatophyte identification techniques.15 The current study's objective is to utilize RAPD-PCR method to identify and distinguish between the strains of fungi present in clinical isolates that cause chronic recurrent dermatophytosis.\n\n\nProtocol\n\nCase control study (observational study). Study participants: 100 participants having skin lesions, hair and nail positive for dermatophytic infection will be included in the study.\n\n\n\n• Samples (Skin scrapings, hair, nail clipping) will be collected from the patients visiting Dermatology OPD, Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe), Wardha.\n\n• Samples will be processed in the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha.\n\n• Method of selection of participants:\n\n1. Patients having skin lesions will be included for taking the skin scrapings.\n\n2. Patients with hair infections positive for fungal infection will be included for hair plucking.\n\n3. Patients with nail infection will be considered for nail clippings.\n\nAll the samples will be collected in a black paper and stored in sterile container with proper labeling for further process.\n\nPatients having visible lesions and itching, positive for fungal infections are included in the study.\n\nSamples will be collected directly from the patients and reports will be obtained from the microbiology laboratory. Data of all patients will be entered in Microsoft Excel taking proper precautions for wrapping the patient identifier information. The final deidentified data will be shared with statistician for further analysis.\n\nSensitivity formula for calculating the sample size.\n\nAlpha (α) 0.05\n\nEstimated sensitivity (Sens) 0.95\n\nPrevalence of disease (Prev) 0.79\n\nEstimated error (d) 0.05\n\nMinimum number of diseases needed: 73\n\nMinimum total sample size needed: 93\n\n\n\n1. Culture media Sabouraud Dextrose Agar (SDA)\n\n2. Culture media Sabouraud Chloramphenicol + Cycloheximide Agar\n\n3. Culture media Dermatophyte Agar Base\n\n4. Dermato Supplement\n\n5. Culture Media Corn meal agar\n\n6. Distilled Water\n\n7. Potassium Hydroxide Pellets\n\n8. Normal Saline\n\n9. 10% Glycerol\n\n10. Lactophenol Cotton Blue Stain\n\n11. Polyvinyl Alcohol\n\n12. Ethyl Alcohol\n\n13. Sodium Hypochlorite Solution\n\n14. dNTP mix, 40mM\n\n15. Hi-Temp PCR Master mix\n\n16. Primers\n\n17. AllPrep® Fungal DNA isolation kit\n\n18. PCR Block Plates\n\n19. Pipette\n\n20. Pipette tips (10μl, 20μl, 100μl, 1000μl)\n\n21. Microscopic glass slides\n\n22. Coverslip\n\n23. Inoculating loop\n\n24. Teasing needle\n\n25. Petri dishes\n\n26. Forceps\n\n27. Glass Beakers\n\n28. Flasks\n\n29. Glass rod\n\n30. Bunsen Burner\n\n31. Gel electrophoresis chamber\n\n\n\n1. Light Microscope\n\n2. Autoclave\n\n3. PCR\n\n4. Biosafety Cabinet\n\n5. Biological Oxygen Demand (BOD) Incubator\n\n6. Electronic Weighing Machine\n\n\n\nA. Sample collection\n\n1. The area of sample collection will be cleaned with 70% alcohol.\n\n2. For skin samples, the lesion will be scraped around the corners using a scalpel blade or glass slide.\n\n3. For nail samples, the nail clippings will be collected using a nail cutter in a sterile container\n\n4. For hair samples, the hair will be plucked from the shaft of the hair having a lesion.\n\nB. Sample processing\n\n1. The skin scrapings will be dissolved in 10% KOH and nail clippings will be dissolved in 40% KOH for microscopic observation.\n\n2. Samples will be inoculated on Dermatophyte Test Medium (DTM) as well as Sabouraud's dextrose agar (SDA) containing chloramphenicol and cycloheximide.\n\n3. Samples will be incubated in BOD incubator, for observation of growth on the SDA and DTM slants.\n\n4. Lactophenol cotton blue staining and slide culture technique will be used to view morphology and colony characteristics of the samples after growth.\n\nC. Application of RAPD-PCR method for molecular characterization\n\n1. Standardization of molecular assay will be done using the standard strains of Trichophyton (D15P127, CBS 118892, UCMS-IGIB-CI11), Microsporum (ATCC 36299), and laboratory-confirmed strains.\n\n2. AllPrep® Fungal DNA isolation kit will be used for DNA isolation from fungal cultures.\n\n3. The primer required for the RAPD-PCR reaction will be synthesized by Beacon designer probe/primer designer software from GeneX India Biosciences Velachery, Chennai, Tamil Nadu.\n\n4. PCR assay mixture, reaction buffer, dNTPs, each primer set (GACA4) and novel primer (CTGT3), DNA template, using these PCR reaction cycles will be carried out.\n\n(39 cycles)\n\na) Denaturation at 93° - 1-minute,\n\nb) Annealing step at 50° -1-minute,\n\nc) -Extension step at 72°- 1 minute\n\nd) Final-extension step at 72°- 7 min\n\n5. Final PCR products will be separated in 0.5X (Tris Borate-Ethylene diamine tetraacetic acid) Buffer and 1% Agarose and stained with the Ethidium-Bromide solution and then the image will be obtained.\n\n6. Interspecies and intraspecies patterns and polymorphism for known strains will be studied.\n\nAll the results will be calculated using R version 4.3.2. Patients enrolled in the study sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) will be resulted from molecular methods used in comparison to the conventional method (gold standard).\n\nCategorical variable will be summarized by the samples positive for fungal hyphae in KOH and culture on SDA (Sabouraud Dextrose Agar) and Dermatophyte Test Medium (DTM).\n\nThe percentage of agreement (overall, positive, and negative) between the two methods will be calculated using agreement analysis (primary and secondary endpoints), along with the Kappa coefficient, p-value, and 95% confidence interval.\n\nDissemination\n\nPapers will be presented at relevant conferences and related studies will be published in indexed journals.\n\nThis research is ongoing. Using previously used primers, we are validating the protocol. The instruments, reagents, enzymes, and primers are set up. Clinical sample collection and processing of the sample by conventional method and standardization of molecular methods for the identification of species is ongoing.\n\nThis study has been granted by the ethics committee of Datta Meghe Institute of Higher Education and Research, Sawangi Meghe, Wardha with the approval number: DMIMS (DU)/IEC/2022/851, dated: 05/04/2022.\n\nWritten informed consent will be obtained from the study participants for participation in the study and publication of their data.\n\n\nDiscussion\n\nFungal infections in humans are becoming more common, especially in immunocompromised people, which has made them a global public health concern. The course of the disease, which can range from mild cutaneous or subcutaneous infections to invasive, widespread, and potentially fatal infections, is determined by the immunological health of the host.16 For epidemiological reasons, accurate antifungal treatment prevention of transmission includes exact separation between dermatophytosis and non-dermatophyte, and thorough identification of disease-causing organisms is vital.17,18 The most reliable approach to diagnose dermatophytosis is through the isolation and identification of dermatophytes from clinical samples. However, it typically takes a long time for the dermatophyte to grow in culture and sporulate, which delays diagnosis. Successful management of dermatophytes depends on prompt diagnosis and correct identification.19 A study from Sweden by Ovrén, E et al. (2016) stated fluorescent staining method enhances the sensitivity and specificity in direct microscopy from skin, hair and nail samples and found that the specificity = (91.7–93.8%), positive predictive value (PPV) = (77.1–81.4%) and negative predictive value (NPV) = (83.7–89.9%).20 Molecular methods are available for the characterization of the dermatophyte species namely restriction fragment length polymorphism (RFLP), (random amplified polymorphic DNA (RAPD), gene-specific-PCR,21,22 chitin synthase encoding gene,23 DNA hybridization,24 and sequencing of the internal transcribed spacer region (ITS).7,25 A study by Li, H. C., Bouchara et al. (2007) from the United Kingdom stated the use of oligonucleotide array based on ITS-1 and ITS-2 sequence for identification of 17-dermatophyte species subsequently, hybridization of a series of oligonucleotides (17–30mers) digoxigenin-labeled PCR products immobilized on a nylon membrane of the 198 clinical dermal filamentous strains tested and 90 non-targeted strains, the array sensitivity and specificity were 99.5% and 97.8%, respectively.26 Many molecular methods have made a way for early detection of dermatophytes to the species level. Along with these techniques serological methods have also detected the dermatophyte infection in the studies stated. A study by Higashi, Y et al. (2012) from Japan stated that the use of newly developed immunochromatographic strip-test for the diagnosis of dermatophytes found the overall sensitivity and specificity of the immunochromatographic test were 83.5% and 66.7%.27", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nWe would like to acknowledge Department of Microbiology, JNMC and Central Research Laboratory (CRL) for their support.\n\n\nReferences\n\nDiongue K, Boye A, Bréchard L, et al.: Dermatophytic mycetoma of the scalp due to an atypical strain of Microsporum audouinii identified by MALDI-TOF MS and ITS sequencing. J. Mycol. Médicale. 2019 Jun; 29(2): 185–188. PubMed Abstract \| Publisher Full Text\n\nWeitzman I, Summerbell RC: The dermatophytes. Clin. Microbiol. Rev. 1995 Apr; 8(2): 240–259. Publisher Full Text\n\nHavlickova B, Czaika VA, Friedrich M: Epidemiological trends in skin mycoses worldwide. Mycoses. 2008 Sep; 51(Suppl 4): 2–15. PubMed Abstract \| Publisher Full Text\n\nGnat S, Łagowski D, Nowakiewicz A, et al.: Infection of Trichophyton verrucosum in cattle breeders, Poland: A 40-year retrospective study on the genomic variability of strains. Mycoses. 2018 Sep; 61(9): 681–690. Publisher Full Text\n\nTaplin D: Dermatophytosis in Vietnam. Cutis. 2001 May; 67(5 Suppl): 19–20. PubMed Abstract\n\nIorio R, Cafarchia C, Capelli G, et al.: Dermatophytoses in cats and humans in central Italy: epidemiological aspects. Mycoses. 2007 Nov; 50(6): 491–495. PubMed Abstract \| Publisher Full Text\n\nGarg J, Tilak R, Singh S, et al.: Evaluation of Pan-Dermatophyte Nested PCR in Diagnosis of Onychomycosis. J. Clin. Microbiol. 2007 Oct; 45(10): 3443–3445. Publisher Full Text\n\nAggarwal R, Targhotra M, Sahoo PK, et al.: Onychomycosis: Novel strategies for treatment. J. Drug. Deliv. Sci. Technol. 2020 Jun; 57: 101774. Publisher Full Text\n\nVerrier J, Monod M: Diagnosis of Dermatophytosis Using Molecular Biology. Mycopathologia. 2017 Feb; 182(1–2): 193–202. Publisher Full Text\n\nBergman A, Heimer D, Kondori N, et al.: Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR. Clin. Microbiol. Infect. 2013 Apr; 19(4): E205–E211. PubMed Abstract \| Publisher Full Text\n\nKondori N, Abrahamsson AL, Ataollahy N, et al.: Comparison of a new commercial test, Dermatophyte-PCR kit, with conventional methods for rapid detection and identification of Trichophyton rubrum in nail specimens. Med. Mycol. 2010 Nov; 48(7): 1005–1008. Publisher Full Text\n\nPaugam A, L’ollivier C, Viguié C, et al.: Comparison of real-time PCR with conventional methods to detect dermatophytes in samples from patients with suspected dermatophytosis. J. Microbiol. Methods. 2013 Nov; 95(2): 218–222. PubMed Abstract \| Publisher Full Text\n\nTartor YH, Abo Hashem ME, Enany S: Towards a rapid identification and a novel proteomic analysis for dermatophytes from human and animal dermatophytosis. Mycoses. 2019 Dec; 62(12): 1116–1126. PubMed Abstract \| Publisher Full Text\n\nBegum J, Mir NA, Lingaraju MC, et al.: Recent advances in the diagnosis of dermatophytosis. J. Basic Microbiol. 2020 Apr; 60(4): 293–303. Publisher Full Text\n\nGräser Y, Fröhlich J, Presber W, et al.: Microsatellite markers reveal geographic population differentiation in Trichophyton rubrum. J. Med. Microbiol. 2007 Aug; 56(Pt 8): 1058–1065. Publisher Full Text\n\nKöhler JR, Hube B, Puccia R, et al.: Fungi that Infect Humans.Heitman J, Howlett BJ, editors. Microbiol Spectr. 2017 May 19; 5(3): 5.3.08. Publisher Full Text\n\nPanasiti V, Devirgiliis V, Borroni RG, et al.: Epidemiology of dermatophytic infections in Rome, Italy: a retrospective study from 2002 to 2004. Med. Mycol. 2007 Jan; 45(1): 57–60. Publisher Full Text\n\nAghamirian MR, Ghiasian SA: Dermatophytoses in outpatients attending the Dermatology Center of Avicenna Hospital in Qazvin, Iran. Mycoses. 2008 Mar; 51(2): 155–160. Publisher Full Text\n\nLiu D, Coloe S, Baird R, et al.: Application of PCR to the identification of dermatophyte fungi. J. Med. Microbiol. 2000 Jun 1; 49(6): 493–497. PubMed Abstract \| Publisher Full Text\n\nOvrén E, Berglund L, Nordlind K, et al.: Dermatophytosis: fluorostaining enhances speed and sensitivity in direct microscopy of skin, nail and hair specimens from dermatology outpatients. Mycoses. 2016 Jul; 59(7): 436–441. Publisher Full Text\n\nKano R, Okabayashi K, Nakamura Y, et al.: Differences among chitin synthase 1 gene sequences in Trichophyton rubrum and T. violaceum. Med. Mycol. 2000 Jan; 38(1): 47–50. PubMed Abstract \| Publisher Full Text\n\nEl Fari, Tietz, Presber, et al.: Development of an oligonucleotide probe specific for Trichophytonrubrum: SPECIFIC OLIGONUCLEOTIDE PROBE FOR T. RUBRUM. Br. J. Dermatol. 1999 Aug; 141(2): 240–245.\n\nKaszubiak A, Klein S, de Hoog GS , et al.: Population structure and evolutionary origins of Microsporum canis, M. ferrugineum and M. audouinii. Infect. Genet. Evol. 2004 Sep; 4(3): 179–186. Publisher Full Text\n\nSharma R, Rajak RC, Pandey AK, et al.: Internal Transcribed Spacer (ITS) of rDNA of appendaged and non-appendaged strains of Microsporum gypseum reveals Microsporum appendiculatum as its synonym. Antonie Van Leeuwenhoek. 2006 Apr 24; 89(1): 197–202. PubMed Abstract \| Publisher Full Text\n\nRippon JW: Medical mycology: the pathogenic fungi and the pathogenic actinomycetes. 2nd ed. Philadelphia: Saunders; 1982; 842.\n\nLi HC, Bouchara JP, Hsu MML, et al.: Identification of Dermatophytes by an Oligonucleotide Array. J. Clin. Microbiol. 2007 Oct; 45(10): 3160–3166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHigashi Y, Miyoshi H, Takeda K, et al.: Evaluation of a newly-developed immunochromatography strip test for diagnosing dermatophytosis: Immunochromatography for diagnosis of dermatophytosis. Int. J. Dermatol. 2012 Apr; 51(4): 406–409. PubMed Abstract \| Publisher Full Text" }	[ { "id": "251787", "date": "27 Mar 2024", "name": "Hassan Aboul-Ella", "expertise": [ "Reviewer Expertise Mycology", "dermatophytosis", "and mycological diagnostic techniques are my major research areas" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 work describes a protocol for an observational study through collecting samples from dermatophytes-suffering patients followed by conventional and molecular diagnostic process, the obtained data will be very useful in improving and updating the epidemiological data in this field.\n\nGeneral comments\nWhile the research focuses on an encouraging area of research and addresses an area of study with minimal existing research, it requires substantial editing of vocabulary in English especially in the following sections; abstract, introduction, and discussion.\nSpecific comments\n- The title needs certain modifications to be more representable for the designed study. Therefore, I suggest \"Diagnosis and molecular characterization of dermatophytosis: an observational study\"\n- The abstract, introduction, and methodological protocol sections are well designed, structured, and described.\n\n- The discussion is missing certain necessary references to support the flow of the work and the researcher point of view in the following points;\n1) The most reliable approach to diagnose dermatophytosis is through the isolation and identification of dermatophytes from clinical samples. However, it typically takes a long time for the dermatophyte to grow in culture and sporulate, which delays diagnosis. - The suggested [Ref 2] in which a culture-dependent cross sectional study has been performed on dermatophytosis showing the importance of culture in dermatophytosis diagnosis as well as the extend of to which extend this technique is laborious and time consuming and that will support the researcher point of view.\n\n2) Many molecular methods have made a way for early detection of dermatophytes to the species level. - The suggested [Ref 3] in which a comprehensive and collective mentioning and illustration of the molecular advancement and molecular diagnostic techniques that is have been developed in the field of dermatophytosis and the extend of the diagnostic level of those techniques and that of course will strongly support the researcher point of view.\n\n3) A study by Higashi, Y et al. (2012) from Japan stated that the use of newly developed immunochromatographic strip-test for the diagnosis of dermatophytes found the overall sensitivity and specificity of the immunochromatographic test were 83.5% and 66.7%. - The suggested [Ref 1] with all respect of the cited study as Higashi et al. 2012, study in the field of immunochromatographic diagnosis of dermatophytosis is really a pioneering study in this field but to be more accurate and more updated I suggest adding the suggested reference in addition to the Higashi et al. 2012, study as the suggested reference represents a newest designed and developed immunochromatographic assay for dermatophytosis diagnosis with a competitive results and certain important production modification over that of Higashi kits.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11929", "date": "08 Jul 2024", "name": "Aditi Warghade", "role": "Author Response", "response": "Respected sir, Thank you for considering our manuscript and suggesting the following changes to be processed for further publication in F1000 research subject to a satisfactory revision as per your suggestions. We have made the changes in our manuscript as per the suggestions given. Please consider the following changes The title needs certain modifications to be more representable for the designed study. Therefore, I suggest \"Diagnosis and molecular characterization of dermatophytosis: an observational study\" Answer: Thank you for the suggestion, sir. I agree with the changes suggested and would like to change the title to \"Diagnosis and Molecular Characterization of Dermatophytosis: An Observational Study\" - The abstract, introduction, and methodological protocol sections are well-designed, structured, and described. - The discussion is missing certain necessary references to support the flow of the work and the researcher's point of view in the following points; 1) The most reliable approach to diagnose dermatophytosis is through the isolation and identification of dermatophytes from clinical samples. However, it typically takes a long time for the dermatophyte to grow in culture and sporulate, which delays diagnosis. - The suggested [Ref 2] in which a culture-dependent cross-sectional study has been performed on dermatophytosis showing the importance of culture in dermatophytosis diagnosis as well as the extend of to which extend this technique is laborious and time consuming and that will support the researcher point of view. Answer: Thank you for the comment, sir. We have added the suggested reference in the draft, quoted as Ref No-4, and highlighted the citation as it is not getting added in the reference list in sequence. 2) Many molecular methods have made a way for early detection of dermatophytes to the species level. - The suggested [Ref 3] in which a comprehensive and collective mentioning and illustration of the molecular advancement and molecular diagnostic techniques that is have been developed in the field of dermatophytosis and the extend of the diagnostic level of those techniques and that of course will strongly support the researcher point of view. Answer: Thank you for the suggestion, sir. We have added the suggested reference in the draft quoted as Ref No- 5, and highlighted the citation as it is not getting added in the reference list in sequence. 3) A study by Higashi, Y et al. (2012) from Japan stated that the use of newly developed immunochromatographic strip-test for the diagnosis of dermatophytes found the overall sensitivity and specificity of the immunochromatographic test were 83.5% and 66.7%. - The suggested [Ref 1] with all respect of the cited study as Higashi et al. 2012, study in the field of immunochromatographic diagnosis of dermatophytosis is really a pioneering study in this field but to be more accurate and more updated I suggest adding the suggested reference in addition to the Higashi et al. 2012, study as the suggested reference represents a newest designed and developed immunochromatographic assay for dermatophytosis diagnosis with a competitive results and certain important production modification over that of Higashi kits.. Answer: Thank you for the suggestion, sir. We have added the suggested reference in our study and quoted it as Ref No-6, and highlighted the citation as it is not getting added in the reference list in sequence." } ] }, { "id": "256151", "date": "14 May 2024", "name": "Saumya Panda", "expertise": [ "Reviewer Expertise Dermatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol, given that there is an epidemic of chronic and recurrent dermatophytosis that is currently ongoing in our country as well as in several other countries and regions in the world. However, the protocol suffers from some serious lapses: 1. The protocol is titled as 'Study of molecular characterization for diagnosis of chronic and recurrent dermatophytosis'. However, the sampling methods and selection criteria of the cases do not mention these conditions at all! 2. Study design is mentioned as 'case control'. However, there is no description of the control group. The description matches that of a cross-sectional study, which is what it should be. 3. In sample size estimation, there is no reference to the prevalence value that has been taken, thus making the whole exercise infructuous. 4. The basic rationale of the study has not been mentioned - the current epidemic of recalcitrant dermatophytosis is due to the emergence of a new species called Trichophyton indotineae, which is morphologically and culturally indistinguishable from Trichohyton mentagrophytes and Trichophyton interdigitale. Thus its identification requires advanced molecular diagnostics like ITS genotyping or modified MALDI-TOF, which are not frequently available.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11930", "date": "08 Jul 2024", "name": "Aditi Warghade", "role": "Author Response", "response": "Respected Madam, Thank you for considering our manuscript and suggesting the following changes to be processed for further publication in F1000 research subject to a satisfactory revision as per your suggestions. We have made the changes in our manuscript. Please consider the following changes. This is an interesting and relevant protocol, given that there is an epidemic of chronic and recurrent dermatophytosis that is currently ongoing in our country as well as in several other countries and regions in the world. However, the protocol suffers from some serious lapses: 1. The protocol is titled as 'Study of molecular characterization for diagnosis of chronic and recurrent dermatophytosis'. However, the sampling methods and selection criteria of the cases do not mention these conditions at all! Answer: Thank you for the suggestion mam. We have changed our title to \"Diagnosis and Molecular Characterization of Dermatophytosis: An Observational Study\". Track changes enabled and highlighted for the same. 2. Study design is mentioned as 'case-control'. However, there is no description of the control group. The description matches that of a cross-sectional study, which is what it should be. Answer: Thank you for the suggestion, mam. We have stated the study design as an observational study as suggested. 3. In sample size estimation, there is no reference to the prevalence value taken, thus making the whole exercise infructuous. Answer: Thank you for the suggestion mam. Mistakenly the study wasn’t added in the prevalence section. I have corrected the draft and added the study with Ref no-3 and highlighted the citation as it is not getting added in the reference list in sequence. 4. The basic rationale of the study has not been mentioned - the current epidemic of recalcitrant dermatophytosis is due to the emergence of a new species called Trichophyton indotineae, which is morphologically and culturally indistinguishable from Trichophyton mentagrophytes and Trichophyton interdigitale. Thus its identification requires advanced molecular diagnostics like ITS genotyping or modified MALDI-TOF, which are not frequently available. Answer: Thank you for the suggestion, mam. I have added the reference information in the draft and quoted it as Ref no 1&2." } ] } ]	1	https://f1000research.com/articles/13-136
https://f1000research.com/articles/13-764/v1	08 Jul 24	{ "type": "Case Report", "title": "Case Report : Navigating the uncommon: Medicopsis romeroi-induced corneal ulcer", "authors": [ "Prerana A Shetty", "Sowjanya Vuyyuru", "Divya Tara", "Sushank Bhalerao", "Prerana A Shetty", "Sowjanya Vuyyuru", "Divya Tara" ], "abstract": "Abstract* This report details a rare case of corneal infection caused by Medicopsis romeroi, highlighting clinical presentation, diagnostic methods, treatment, and outcome. An early adolescence male, after recent corneal tear repair, presented acute eye pain, redness, and decreased vision for 1 day. He underwent corneal tear repair elsewhere post vegetative matter-related swimming injury. Ophthalmic examination revealed a 2.53.3mm ulcer around sutures. Corneal scrapings enabled microscopy and culture. With lesion worsening, tissue adhesive (TA) and bandage contact lens (BCL) were applied. Fungal culture identified Medicopsis romeroi. Patient's history, clinical course, and treatment were documented. Microscopy unveiled fungal hyphae, later confirmed as Medicopsis romeroi. Treatment included topical/systemic antifungals. Post-TA + BCL, significant improvement followed, with re-epithelialization, scarring. This report underscores the importance of considering rare fungal pathogens like Medicopsis romeroi in cases of corneal ulcers, especially in individuals with unusual risk factors. Timely diagnosis through microscopy and fungal culture, coupled with tailored antifungal treatment led to successful management and favorable patient outcomes. This case also emphasizes the significance of vigilant clinical observation, precise laboratory analysis, and multidisciplinary collaboration between ophthalmologists and microbiologists in managing atypical ocular infections.", "keywords": [ "fungal keratitis", "rare fungus", "natamycin", "tissue adhesive", "bandage contact lens" ], "content": "Introduction\n\nFungal keratitis typically results from the external acquisition of fungal agents, usually resulting from the traumatic implantation of fungal fragments or spores into the cornea. Notably, plant debris-related injuries account for a substantial 60% of fungal keratitis cases, making them the predominant causative mechanism. Fungal keratitis can be attributed to a spectrum of cornea-pathogenic fungi. These include species within the genera Fusarium, Aspergillus, Candida, Curvularia, and Penicillium. The prevalence of these fungi may vary based on geographical, occupational, and host-specific factors. Nevertheless, several fungal species are consistently detected as a cause of keratitis.1 An emergent pathogen Medicopsis romeroi is allocated to phylum Ascomycota according to National Center for Biotechnology Information. These organisms are widely distributed in the soil and plants, and they are able to infect people by direct inoculation. It is an emerging fungus causing subcutaneous phaeohyphomycosis. It has mostly been reported in immunocompromised patients receiving immunosuppressive drugs for hematologic malignancy, renal transplantation, rheumatoid arthritis, leprosy, and asthma; however, there has been one report of a healthy, immunocompetent host.2 Comprehensive discussions about Medicopsis romeroi are lacking in existing medical literature. Therefore, this case report aims to shed light on the clinical features and management strategies employed in a case of corneal ulcer caused by Medicopsis romeroi.\n\n\nCase report\n\nA male in his late teens presented to cornea clinic with complaints of redness, pain and decreased vision in his left eye. He had undergone corneal tear repair elsewhere after trauma with vegetative matter while swimming. At the time of presentation, his visual acuity was 20/20 in right eye and counting finger at 1 metre in the left eye. Upon detailed examination using slit lamp biomicroscopy, we observed a sutured corneal tear which was surrounded by a 2.53.3mm full thickness corneal infiltrate. [Figure. 1a] In order to assess the status of the posterior segment, a B-scan ultrasound was conducted, which showed no evidence of posterior segment involvement.\n\nCorneal scraping was done, and the obtained material was then prepared with potassium hydroxide (KOH) for microscopic examination, which unveiled septate fungal filaments. The patient was treated with natamycin 5% eyedrops, administered hourly, coupled with oral ketoconazole 200mg twice a day and cycloplegics. Further the material was cultured but did not grow and organisms. Worsening was noted with the infiltrate increasing in size and severity and anterior chamber (AC) leak was noted. [Figure 1b] Repeat corneal scraping with AC reformation, TA and BCL was done. Topical Voriconazole 1% drops was added to the treatment. This time the culture revealed significant growth of dematiaceous fungi which was later identified as medicopsis romeroi. Over four months, there was significant improvement with scar formation with vascularization. [Figure. 2] Visual acuity also improved to 20/80. Corneal topography followed by contact lens trial was conducted, and the patient accepted the lens, improving vision to 20/100. [Figure 3a] Anterior segment OCT was done which revealed dense stromal scar tissue along the full thickness and he also developed an anterior capsular opacification in the visual axis. [Figure 3b] Synechiolysis was performed to release the anterior synechiae formed. The visual acuity improved to 20/50 and in view of zonular dialysis, pars plana lensectomy was planned.\n\n\nDiscussion\n\nIn the presented case, our patient was diagnosed with fungal keratitis caused by Medicopsis romeroi, highlighting the persistent challenge of fungal keratitis as a leading cause of visual impairment in developing nations. An understanding of the regional epidemiological features, risk factors, and etiological agents is important in the prevention and appropriate management of this disease entity.3 Fungal keratitis manifests in two primary presentations: filamentous fungal keratitis and yeast-like fungal keratitis. Filamentous fungal keratitis encompasses a spectrum of potential pathogens, including Fusarium, Aspergillus, Curvularia, Scedosporium apiospermum, Phaeohyphomycetes, and Paecilomyces. Identified risk factors for fungal keratitis encompass corneal trauma, contamination with plant material, immunosuppression, contact lens-related trauma, corneal surgery, and chronic keratitis. Diagnosis of fungal keratitis is traditionally established through culture techniques involving blood agar, Sabouraud agar, or brain–heart infusion broth, with corneal biopsy yielding a positive organism isolation rate of up to 82%.4\n\nDematiaceous fungi, also referred to as melanized fungi, derive their dark pigmentation from the presence of melanin within the hyphal cell wall. More than 150 species and 70 genera of dematiaceous fungi have been implicated in human or animal diseases, and the list is expanding due to advances in the techniques applied to modern taxonomy. Dematiaceous fungi are primarily saprophytic, commonly inhabiting soil and plant environments. While they infrequently cause clinical disease, their significance as pathogens becomes pronounced, particularly in immunocompromised individuals.5 M. romeroi (formerly Pyrenochaeta romeroi), belongs to the saprophytic dematiaceous fungi and is classified within the order Pleosporales and the class Coelomycetes. These filamentous fungi are distinguished by their production of conidia within specialized fruiting structures called pycnidia. Coelomycetes are known to cause various superficial or subcutaneous infections, including onychomycosis, subcutaneous phaeohyphomycosis (PHM), and eumycetoma. They exhibit a wide geographic distribution, commonly thriving in soil and plants, particularly in tropical and subtropical regions, with the capacity to infect humans through direct inoculation into the skin. Human cases of M. romeroi infections are rare and primarily manifest as subcutaneous PHM or black grain eumycetoma.6\n\nRegardless of the clinical manifestation, the most common approach to management of M. romeroi infection was treatment with azole antifungals along with surgical excision. The challenge lies in the fact that most azole antifungals have shown a relatively high failure rate in the face of M. romeroi. It is unclear which antifungal is the drug of choice, whereas the low reported minimum inhibitor concentrations and favourable side effect profile of voriconazole support its use against M. romeroi7 In the secondary analysis of the mycotic ulcer treatment trial II, the presence of hypopyon at baseline was associated with a 2.3-fold increase in the odds ratio of needing TPK. However, our patient did not present with hypopyon, neither at diagnosis nor at follow-up, and, no surgical procedure except TA+BCL with AC reformation was necessary for the resolution of the fungal infection, demonstrating inactivity after four months of follow-up.1,8\n\n\nConsent\n\nWritten informed consent for the publication of the case report and any associated images was obtained from the patient prior to submission.\n\n\nEthics and consent\n\nEthical approval and consent were not required.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nNone\n\n\nReferences\n\nSáenz-Madrazo N, Baeza A, Guinea J, et al.: First report of neocucurbitaria unguis-hominis keratitis. Journal of Fungi. 2022; 9: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChanyachailert P, Leeyaphan C, Bunyaratavej S, et al.: Subcutaneous phaeohyphomycosis from Medicopsis Romeroi in a diabetic patient. Medical Mycology Case Reports. 2019; 26: 69–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGopinathan U, Garg P, Fernandes M, et al.: The epidemiological features and laboratory results of fungal keratitis. Cornea. 2002; 21: 555–559. Publisher Full Text\n\nLakshmi S, Garcia CV: Recalcitrant Paecilomyces keratitis. BMJ Case Rep. 2019; 12: e229226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLos-Arcos I, Royuela M, Martín-Gómez MT, et al.: Phaeohyphomycosis caused by medicopsis romeroi in solid organ transplant recipients: Report of two cases and comprehensive review of the literature. Transpl. Infect. Dis. 2019; 21: e13072. PubMed Abstract \| Publisher Full Text\n\nAljundi M, Brun S, Akhoundi M, et al.: Recurrent subcutaneous phaeohyphomycosis due to Medicopsis romeroi: A case report in a dermatomyositis patient and review of the literature. Microorganisms. 2022; 11: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarofalo F, Wilcock J, Lahey T: Posttraumatic endophthalmitis caused by Medicopsis Romeroi. Infect. Dis. Clin. Pract. 2022; 30. Publisher Full Text\n\nPrajna NV, Krishnan T, Rajaraman R, et al.: Predictors of Corneal Perforation or Need for Therapeutic Keratoplasty in Severe Fungal Keratitis: A Secondary Analysis of the Mycotic Ulcer Treatment Trial II. JAMA Ophthalmol. 2017; 135: 987–991. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "310014", "date": "09 Sep 2024", "name": "Alaa Atef Ghaith", "expertise": [ "Reviewer Expertise I am a cornea and external eye disease consultant with expertise in the medical and surgical management of corneal disorders. I am a professor of Ophthalmology at the faculty of Medicine - Alexandria University - Egypt. I have a long history in the management of infectious keratitis in this facility and a number of publications in this field" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 importance stems from the rarity of this fungal keratitis (Medicopis romeroi). It is an addition to our knowledge of new fungal species that can infect the cornea. The management of the case using tissue adhesive and bandage contact lens upon leakage from the AC is controversial as it is not advised to place a contact lens on an infected corneal lesion. Also, the final photo of the corneal vascularized scar after 4 months is not correlated with the visual acuity of 20/80. However, the case was presented in sufficient details regarding the history, clinical findings, progress, treatment and final outcome.\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": "317902", "date": "24 Sep 2024", "name": "Savitri Sharma", "expertise": [ "Reviewer Expertise Ocular Microbiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report describes a rare cause of fungal keratitis-Medicopsis romeroi. While the clinical descriptions are well presented and well documented, the fungal characteristics are missing. Laboratory processing of the corneal scrapings is poorly described and the reader does not get to learn the fungal characteristics that were used to identify the fungus nor is there any confirmatory test done. Some of the expressions are unusual, for example \"Microscopy unveiled ...........romeroi \" in line 8 in abstract. It is not clear whether it refers to initial microscopy of corneal scraping or microscopy of culture isolate for identification. Introduction requires references for some of the statements. All organisms must be written in italics. Photo-documentation of the corneal scraping microscopy as well as culture are required towards evidence supporting the diagnosis of the case. Language needs greater care. The line 11 sentence- \"Further..........organisms \" in case report is incomplete that conveys no meaning. It is not clear how the second sample was processed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/13-764
https://f1000research.com/articles/13-763/v1	08 Jul 24	{ "type": "Research Article", "title": "Proposing an Effective Waqf Model for Ensuring Poverty Reduction among Refugees in Malaysia", "authors": [ "Adnan Opeyemi Salaudeen" ], "abstract": "Background COVID-19 has heavily impacted the financial and economic well-being of the world’s population. Various national relief programs and initiatives have been carried out to address society’s financial difficulties, especially the issue of poverty among the vulnerable, like refugees and immigrants. Consequently, being one of the main elements in Islamic social finance, waqf has been emphasised and proven throughout Islamic history as one of the practical tools for poverty reduction. However, recent waqf initiatives towards poverty reduction focused more on aiding the micro and small entrepreneurs, whilst limited waqf initiatives were available to help low-income households, refugees, and immigrants during this critical time in Malaysia.\n\nMethods This study introduces a novel waqf model, specifically designed to contribute to poverty reduction in society. It is tailored for low-income households, refugees, and immigrants, aiming to sustain their financial independence. By combining the elements of waqf and crowdfunding, this model proposes an innovative agriculture-based microfinance programme. It utilizes undeveloped waqf lands and equity-based crowdfunding to reach its objective of poverty reduction. This study has adopted a secondary research approach using the existing scholarly literature.\n\nResults This paper also discusses the potential of the proposed model to deliver more than its primary objective, as the framework illustrates that waqf institutions will have a continuous inflow of funds that will allow them to initiate other waqf-based projects, job creation through the microfinance programmes, and financial return for the investor of equity-based crowdfunding projects.\n\nConclusion This study not only underscores the sustainability of the proposed model, but also its alignment with the increasing awareness of value-based and social impact objectives. It further explores the potential of waqf to be integrated with other financing instruments during the pandemic to help the vulnerable, specifically refugees and immigrants. This research thus contributes to the true purpose of Islamic social finance, highlighting the significance of its findings.", "keywords": [ "Covid-19", "Waqf", "Crowdfunding", "Microfinance", "Malaysia" ], "content": "1 Introduction\n\nIn October 2020, the World Bank (2020), in its press release, estimated that an additional 88 million to 115 million people would be pushed into extreme poverty, resulting in a total estimation of 150 million in 2021, all due to the economic impact of the recent global pandemic of COVID-19. Poverty during COVID-19 is a worrying issue that affects all countries globally, mainly due to the emerging trend of unemployment faced by many throughout this current situation. Banerjee and Duflo (2011), in their paper, stated that technically, increasing poverty and declining income in society would affect more than just one’s financial independence, as it can result in highly damaging impacts on one’s condition of life in the long run, including health quality, and access to good education and nutrition. Furthermore, following the statement mentioned earlier, the International Monetary Fund [IMF] (2020) has stated that the movement of households into poverty during this time has damaging effects on an individual’s economic mobility.\n\nTherefore, poverty eradication and reduction have been the top priority as it was placed as the first goal in UNSDG (World Bank Group 2020). Even from an Islamic perspective, Sadeq (1997) stated poverty is a form of social evil, as it degrades the human’s status as vicegerent of Allah and has an endogenous impact on social problems. Waqf, which falls under a philanthropy-based institution, has been acknowledged as one of the essential philanthropy-based instruments throughout the evolution of Islamic finance over the past decades (Nazrul et al., 2017). This can be seen as addressing the issue of poverty, was constituted one of the main objectives of awqaf since Islamic history, as well as the development of socio-economic inclusion through waqf, when the first philanthropic Waqf of Islamic history traced back to the years of caliph Omar R. A (Sadeq, 1997).\n\nAgain, people are being uprooted and forced to escape for their safety in many different places of the world. According to the United Nations High Commissioner for Refugees (UNHCR, 2019), at least 70 million individuals were reported to have been forcibly displaced in 2018. Many more are believed to be missing. Most people flee life-threatening danger or persecution in favour of education, better careers, or better lives (Mohamad et al., 2021). These people seek protection and shelter abroad due to conflicts or open discrimination based on religion, ethnicity, or even political ideas and affiliations. Instruments like microfinance and waqf can be helpful for these hapless people. However, refugees are frequently excluded from these microfinance programmes, although the development actors consider microcredit and microsavings to be ways to safeguard and combat poverty among the poor.\n\n\n2 Methodology\n\nAs the issue of poverty becomes more severe due to the recent global pandemic, more innovative waqf instruments and initiatives must be implemented to curb and contribute continuous efforts towards poverty alleviation. Therefore, this paper aims to discuss the proposed waqf model in helping the poor and reducing the poverty rate. This research project has utilized a secondary research approach by drawing on existing scholarly literature to gather relevant information and insights. The paper is organised as follows: Section II discusses some literature on utilising undeveloped waqf lands, microfinance and poverty. Section III discusses the proposed waqf and crowdfunding-based microfinance model and the related elements involved. Section IV analyses the model’s operation’s strengths, weaknesses, opportunities and threats, practicality, risks, arising issues, do-ability, and sustainability. The last section analyses the opportunities to implement this model in the EU, followed by a conclusion, concluding the whole paper (Rapi et al., 2022).\n\n\n3 Literature review\n\nPoverty is known as one of the primary challenges combating humanity. One of the most critical missions of the United Nations Sustainable Development Goals (SDG), founded in 2010, is to eliminate poverty by 2030. Poverty is characterised as a situation in which a community or a segment of the population can only meet their basic needs, such as food, clothing, and shelter, to maintain a minimum standard of living (Mohr, 2016). The Global Monitoring Report (2009) defined poverty as the population living on less than $1.25 daily. According to the World Bank (2019), two billion eight hundred people (2.8 billion), or nearly half of the world’s population, live on less than $2 per day, and 1.2 billion live on less than $1 per day. Addae-korankye (2014) elaborates on the causes of poverty as unemployment: the absence of any economic activity that could provide a steady flow of profits. Vulnerability to Income Fluctuations: Many households experience income fluctuations and may have to go to bed without food for family members. Powerlessness is the inability to change anything or do something to improve the situation.\n\nAccording to Srinivasan and Sriram (2003), microfinance involves providing financial services to small and medium enterprises that lack access to banking and related services due to high transaction costs and qualifying factors. Seibel (2003) outlines the following priorities for microfinance institutions (MFIs) in developing countries: 1. Alleviating extreme poverty and hunger 2. Achieving universal primary education 3. Promoting gender equality and women’s empowerment 4. Lowering infant mortality 5. Improving maternal health 6. Establishing income-generating activities (creating jobs) 7. Acquiring properties 8. Consumption stabilization 9. Risk protection 10. Encouragement of new businesses 11. Developing a strategy to build global financial systems that meet the needs of the poorest people. Numerous case studies demonstrate how microfinance has contributed to eradicating poverty. It primarily achieves this through:a) Microcredit b) Micro Savings c) Micro-Insurance.\n\nThe emergence of the Microfinance Movement has altered perspectives on supporting impoverished individuals in Asia and Latin America. Many countries have issued noteworthy loans, particularly to low-income families or individuals who would typically be overlooked by formal financial institutions (Nazrie Mohd Nor and Kumar, 2019). According to Md Saad (2012), the microfinance objectives set at the 1997 Microcredit Summit have largely remained unmet. The very poorest are the least likely to benefit from microfinance, and despite the availability of microfinance, there has been no improvement in wages. A significant portion of the loans is used for consumption, with the remaining funds earmarked for quickly-planned projects. Nevertheless, there have been notable benefits, such as consumption smoothing and the creation of social networks, which can be equally important. The claim about fiscal sustainability argues that for-profit MFIs would be able to reach a larger number of impoverished people. Equity-Based Crowdfunding\n\nEquity crowdfunding, also known as crowd-investing or investment crowdfunding, is a method used by startups and early-stage businesses to raise capital. It involves offering the company's securities to potential investors in exchange for funding. Each investor receives a proportionate share of the company based on their investment. Unlike rewards or donation crowdfunding, equity crowdfunding offers a more traditional method of raising capital by providing financial securities to investors (Di Pietro, 2020).\n\nCrowdfunding is conducted using specialised online platforms like Wefunder and StartEngine. The digital aspect of the crowdfunding platform promotes a more permissive and open approach to funding (Nehme, 2018). As a result, equity crowdfunding appeals to a broader audience, unlike traditional capital-raising approaches for early-stage firms, which rely mainly on contributions from a small community of professional investors. The primary goal of equity crowdfunding is to collect the necessary money by soliciting small investments from many investors (Di Pietro, 2020). Malaysian ECF platforms have grown significantly in response to the government’s call for financial service providers to adopt technology to create a more inclusive, creative, and competitive capital market. As of December 2019, investments totalled RM73.74 million, with 80 successful campaigns and 77 successful issuers. According to the investment demographic, 46 percent of participants were under the age of 35, and 52 percent of the investment came from the retail sector (Ibrahim et al., 2018).\n\nAs Htay et al. (2015) highlight, many researchers and academicians vibrantly advocate for more robust development towards endowments and waqf to ensure the welfare of the Muslim ummah and society as a whole. Furthermore, as the topic and effort on poverty reduction being emphasised by a significant amount of academic community from different backgrounds, Saiti et al. (2020) highlighted that this issue still continuously affects millions of Muslims whilst urging for further development of waqf that could be contributing to the real potential towards poverty reduction and alleviation. Based on the literature reviews in the sections above, this study attempts to develop an effective model for addressing the issue of poverty in Waqf’s body of knowledge (Olalekan and Hakeem, 2012). Remarkably, the model attempts to corporate elements of waqf through utilising undeveloped waqf land and involvement of Islamic microfinance institutions and crowdfunding platforms in agriculture-based microfinance programmes.\n\nMalaysia is recognised for granting asylum until their return or relocation to another nation. According to reports, Malaysia had the most refugees in Southeast Asia as of January 2018 (UNHCR, 2019). There were 178,140 refugees and asylum seekers in Malaysia as of January 2021. The bulk of them lived in Selangor (66,030), Kuala Lumpur (27,370), and Penang (18,660), while the least number lived in Putrajaya (450) and Perlis (280) (Mohamad et al., 2021). More than half of these people are Rohingya refugees who hail from Myanmar (Mohamad et al., 2021). Programmes based on waqf and microfinance may be crucial for this sizable population of immigrants and refugees.\n\nAccording to Wahab (2017), even though refugees work difficult occupations to make a living, they only receive poor pay. Some of them make less than the RM1000 minimum wage that the Malaysian government established in 2016. Wahab (2017) also found that some self-employed refugees rented trading licenses from local traders to operate their businesses since they are not legally permitted to apply for licenses to own businesses. Other self-employed refugees received financial assistance from microfinance institutions or family and friends to set up their businesses. Then, these independent refugees assist their fellow refugees by providing them with employment and products (Wahab, 2017). The economic and financial difficulties they experienced have resulted in a number of other problems, namely in health and education (Mohamad et al., 2021).\n\n\n4 Findings and discussion\n\nThe waqf and crowdfunding-based microfinance model incorporates waqf and crowdfunding elements in the microfinance programme. The waqf element utilised in this proposed model is undeveloped waqf lands abundant in Malaysia. This can be seen as a study by Mahmood et al. (2017) highlighted that Yayasan Wakaf Malaysia (YWM) estimated that there are around 30,889 hectares of waqf lands in Malaysia, whilst a paper (Mohammad, 2009) has reported that around 90% of the waqf lands are still undeveloped (Mohd et al., 2020). Therefore, sources of funds to carry out this programme come from two sources, which are (i) undeveloped waqf lands under the management of waqf institutions, and (ii) collected funds from a crowdfunding platform which is categorised as equity-based crowdfunding. Islamic microfinance institutions will act as intermediaries in channelling the undeveloped waqf lands and collecting funds to carry out the Waqf-based Microfinance Programme.\n\ni- Waqf Principles\n\nIn order to ensure the proposed model is waqf-based, it must fulfil the four-principle elements of waqf, which are (i) donor (waqif), (ii) waqf corpus (mawquf), (iii) beneficiaries (mawquf alayh), and (iv) manager (mutawalli/nazir) (Mohamad et al., 2016). In this model, the waqif will be the donor of undeveloped waqf lands, the waqf corpus undeniably will be the abundance of undeveloped waqf lands, beneficiaries of the waqf will be the people who live in poverty nearby the waqf lands, whilst the nazir/mutawalli will be the State Islamic Religious Council. Waqf is often classified into a few categories: timing, purpose and object. Under the category of waqf object, undeniably undeveloped waqf land falls under immovable assets. For timing, this waqf model sets to be permanent waqf. According to Siti et al. (2016), permanent waqf is defined as utilising the accumulation of waqf assets, producing a continuous flow of revenue to serve its purpose of social impact. This model aims to operate according to the basis mentioned earlier towards poverty reduction. For its creation, this waqf falls under the waqf khas, as it is specifically meant for the poor who live near the undeveloped waqf land. By joining the tailor-made agriculture-based microfinance programme will be carried out by Islamic microfinance institutions through this waqf model (Zakariyah et al., 2022). This further clarifies that the mawquf asset used in this waqf is classified under direct waqf, as the waqf asset directly will be channelled to target beneficiaries towards poverty reduction and help them to reach financial independence through involvement in entrepreneurship (agriculture-based microfinance programme).\n\nii- Undeveloped Waqf Lands (UWL)\n\nUndeveloped waqf land is one of the three main elements that will be channelled in the waqf-based microfinance programme. Being the mutawalli of the undeveloped waqf lands, waqf institutions in this model will channel the waqf lands to the Islamic microfinance institution to be utilised for the waqf-based microfinance programme under the contract of Ijarah. The relevance behind charging on utilisation of the UWL instead of giving it away is (1) to ensure discipline and responsibility of microfinance clients in cultivating and taking care of the waqf land productively and sustainably. (2) Ensuring the regular flow of funds’ towards waqf institutions to allow and enable waqf institutions to contribute to waqf-based projects that will bring positive social impacts to the society. This will potentially result in various social impacts; to the microfinance clients of the programme (poverty reduction) through engaging in entrepreneurship, creation of jobs if the agriculture business expands positively, and to the targeted society that will benefit from other waqf-based projects made by the waqf institutions using utilisation payment of the undeveloped waqf lands (Zakariyah et al., 2023).\n\niii- Parties to Waqf and Crowdfunding-Based Microfinance (WCM)\n\na) Target groups as Beneficiaries (Mawquf ‘Alayh): Poverty is classified under two main categories: displaced and absolute poverty. Due to the recent pandemic hit, the World Bank has stated that it is expected that there will be a rise in new poor, ranging between 119 and 124 million (World Bank, 2021). Therefore, several waqf initiatives are being launched to contribute to poverty alleviation. For example, Santi et al. (2021) studied the cash waqf project towards poverty reduction among micro-entrepreneurs in Gunung Kawi District, Malang Regency, Indonesia. Moreover, the launch of Dompet Dhuafa in Indonesia to assist SMEs using the collection of waqf funds through an online platform, and waqf entrepreneurship towards poverty reduction in Kano, Nigeria (Nura & Asmak, 2020), are some of the waqf initiatives that have been implemented in few countries to address the recently increasing poverty issues. However, these initiatives towards poverty reduction mainly emphasised displaced poverty; people who are initially not living under the poverty line, however, pushed below the poverty line due to a few circumstances such as loss of jobs and bankruptcy due to shutting down of businesses (micro and small entrepreneurs). Through this model, the goal of poverty reduction also covers helping absolute poverty, in which the category of people who live below $1.90 per day to fulfil basic living needs, according to the Global Poverty Line (World Bank, 2020).\n\nThe global refugee crisis has impacted almost every country. However, the government of Malaysia does not formally recognise the legal status of refugees. Hence the current flood of migrants in this country is alarming. This raises various issues, including whether they will be able to survive in Malaysia and whether the current institutions and laws can meet their demands for healthcare, education, and employment opportunities. Despite having fled torture and persecution in their home countries, they would still have to deal with difficulties as refugees in Malaysia. Programmes involving waqf and microfinance can be immensely beneficial for these refugees in Malaysia so that they can get out of the poverty trap and lead a life with dignity and respect (Mohamad et al., 2021).\n\nTherefore, there is a need to emphasise effective strategy towards the targeted groups and people to join this programme, which are micro and small entrepreneurs that experienced business shutdowns and poor people living in absolute poverty near the undeveloped waqf lands. Using land and microfinance, together with sufficient training, will help them grow out of the poverty bubble efficiently and effectively through micro and small agriculture businesses.\n\nb) Islamic Microfinance Institution (IMFI): According to Khanam et al. (2018), microfinance has been infamous for being an efficient tool for entrepreneurs to alleviate and reduce poverty. In this model, waqf-based microfinance programmes that Islamic microfinance institutions will carry out will operate on group lending and utilise social collateral as risk mitigation tools in microfinancing. Several studies made on Amanah Ikhtiar Malaysia (AIM), which adapts the group lending and social collateral in its microfinance programme, have shown to result in a meagre rate of default, high rate of repayments and effective in reducing poverty through microfinance among low-income households, especially among women (Haque et al., 2019). Islamic microfinance institution in the model acts as intermediaries in channelling three main elements of the waqf-based microfinance programme (undeveloped waqf lands, collected funds from the crowdfunding platform, entrepreneurial and agriculture training) to the microfinance clients. Since microfinance institutions like Amanah Ikhtiar Malaysia (AIM) already provide entrepreneurial and financial literacy training to its clients (Engku, 2019), making it is relevant for the chosen Islamic microfinance institutions to provide agricultural and entrepreneurial training in the waqf-based microfinance programme (Salaudeen & Zakariyah, 2022; Salaudeen, et al. 2022).\n\nc) Crowdfunding Platform (Equity-based Crowdfunding): The microfinancing source will be equity-based crowdfunding for the second element of the waqf-based microfinance programme. In this matter, Islamic microfinance institutions will approach crowdfunding platforms to launch equity-based crowdfunding projects, with proof and related legal documentation regarding the waqf-based microfinance programme. Return on the investment made to the programme will be based on a payment-by-success basis, as adopted in the bond market as Social Impact Bonds, which has proven to have several success stories in terms of social impact and return to the investors. Social Impact Bonds (SIB), according to (Marwan and Haneef, 2019), are innovative mechanisms where public, private and voluntary sectors come together to address social issues that focus on delivering outcomes (UK Government, 2012). SIB is a commissioning tool where social investors fund a social project and get returns based on the achieved social impacts and results. In the structure of SIB, payment-by-success is a usual term for return on investments; the project’s success will be measured and reported to the outcome payer (usually the government). This will determine the project’s success, whether the investors will get capital plus a return or risk losing all their investment if their project fails (Marwan & Haneef, 2019).\n\nPotentially, an adaptation of how SIB works and the payment-by-success element can be implemented under this waqf and crowdfunding-based microfinance. This can be seen through one of many success stories of SIB, such as Peterborough SIB, the world’s first SIB that was issued for a social programme project to reduce the re-offending rate of HMP Peterborough’s short-sentenced offenders. Its first programme successfully reduced the re-offending rates, resulting in investors’ getting their return. Most of the parties to this waqf & crowdfunding-based microfinance model are government-related, and utilisation of the crowdfunding platform is in line with the size of the project, which is about providing a microfinance programme through the utilisation of undeveloped waqf lands and microfinancing, all for the purpose to help people to reach financial independence and freedom, as well as reducing poverty. Therefore, emphasising the similarities between the SIB flow and this proposed poverty reduction model aligns with various national development agendas on goals in alleviating poverty (Saad et al., 2023).\n\niv- Contracts Involved\n\nIn waqf and crowdfunding-based microfinance models, at least three central contracts will be applied throughout the whole model; Ijarah, Musharakah and Commodity Murabahah (Tawarruq). First, an Ijarah contract will be made between waqf institutions and Islamic microfinance institutions to utilise the undeveloped waqf lands in waqf-based microfinance programmes. In this matter, payment under the Ijarah contract will be made by IMFI to the waqf institution using weekly repayments made by microfinance clients. The next main contract is a profit-loss sharing arrangement in the form of Mudarabah, made between Islamic microfinance institutions and crowdfunding investors through the crowdfunding platform (Mohd Ali et al., 2015). Since the crowdfunding will be equity-based, IMFI will approach the crowdfunding platform to launch the equity-based crowdfunding project with the clause of payment-by-success, strengthening the relevance behind the utilisation of the Mudarabah contract. Finally, the commodity Murabahah is another main contract that will be utilised under this model in providing microfinance to the microfinance client. In Malaysia, most microfinance institutions provide shariah-based microfinancing using the Commodity Murabahah contract. One of the examples is Amanah Ikhtiar Malaysia (AIM), a government-based microfinance institution that offers micro-financing to its microfinance clients through Tawarruq/Commodity Murabahah arrangement (Engku, 2019). Initially, microfinancing offered by AIM was under the contract of Qard. However, after a review by Shariah panels in 2010, it was made clear that the best contract to be used in AIM microfinancing is Tawarruq due to the 1% management fee imposed by AIM on every microfinancing given (Engku, 2019).\n\nv- Legal and Regulatory Framework\n\na) SIRC: In Malaysia, the State Islamic Religious Council (SIRC) is responsible for handling and managing all matters related to the waqf in accordance with the Ninth Schedule of the Federal Constitution, List II (State List) (Ismail et al., 2015). The SIRC has the authority as the sole trustee of waqf land under each state's enactment. Therefore, the council exclusively manages and administers all waqf land, whether movable or immovable. It has also been specified that the SIRC in every state in Malaysia acts as the sole trustee for all waqf land and properties, including general waqf, special waqf, general Nazr, and all trusts (Ismail et al., 2015).\n\nThe provisions mentioned in Section 63 of the Administration of Islamic Religious Affairs (Terengganu) Enactment, No. 2 of 2001 state that the Majlis shall be the sole trustee of all waqf, Nazar, and charitable trusts for the support and promotion of Islam or the benefit of Muslims under Syariah laws in the State of Terengganu. Section 64 of the same Act states that all property subject to section 63 shall vest in the Majlis for the waqf, Nazar, or trust affecting the property. The appointment of SIRC as the sole trustee of the waqf property aims to ensure proper administration and management to generate profits for charitable activities. It helps to prevent problems that may arise from appointing unreliable independent trustees or from encroachment by heirs that could lead to the loss of waqf land. From a legal perspective, SIRC has the right to manage and develop waqf land in accordance with Islamic principles, including entering agreements, purchasing, owning, exchanging, transferring, and developing waqf land in Malaysia.\n\nb) Crowdfunding Platform: Choo Hong (2018) discussed that Malaysia established a regulatory framework in 2015 to facilitate crowdfunding, and another in 2016 to promote peer-to-peer lending. Malaysia currently has few financial reporting standards and laws for non-equity crowdfunding (ECF) and P2P financing. The Securities Commission of Malaysia (SC) established the Guidelines on Recognized Markets in 2015 and updated it in 2016, which contains most of the regulations governing alternative finance. The main focus of the Recognised Markets Guidelines is the financial reporting aspects of the Recognized Market Operator (RMO) set-up and reporting to the SC. According to paragraph 1.03 of the Guidelines, all RMOs must be a corporation or a limited liability partnership (LLP) and are governed by either the Companies Act 2016 or the Limited Liability Partnerships Act 2012. It is worth noting that unlike in other countries, societies or non-profit organizations, such as those promoted on Kickstart, are not allowed to become RMOs; however, non-profit organizations can become RMOs if registered as corporations or limited liability partnerships. All RMOs must file their latest audited financial information with the SC within three months of the end of each financial year, and comparable filings must be submitted to the CCM if required by the Companies Act 2016 or the Limited Liability Partnerships Act 2012.\n\nvi- Flow of Waqf and Crowdfunding-based Microfinance Model\n\nThe model starts with waqf institutions (mutawalli) channelling the undeveloped waqf lands to the Islamic microfinance institutions, as the institution is the one who will be carrying out the microfinance programme. Being the intermediary, Islamic microfinance institutions will then approach crowdfunding platforms to launch the crowdfunding project for the microfinance programme through equity-based crowdfunding. During the approach, Islamic microfinance institutions will provide the crowdfunding platform with documents that signify the programme’s authenticity, target funds needed, and documentation of principal and return. Once the targeted fund has been collected, the crowdfunding platform will channel the collected funds to the Islamic microfinance institution. It then follows with the carrying out of the waqf-based microfinance project that will be channelling three elements to the targeted and chosen microfinance clients towards this programme, which are (i) undeveloped waqf lands, (ii) microfinancing, and (iii) agriculture and entrepreneurial training.\n\nIn the waqf-based microfinance programme for agriculture, repayments shall be made by microfinance clients according to the norms of Islamic microfinance institutions. Adapting the repayment and risk mitigation techniques applied in the microfinance programme under Amanah Ikhtiar Malaysia (AIM), the programme will be adapting group lending, small weekly repayments during central meetings, and social collateral as risk mitigating techniques in avoiding potential defaults and failure of the programme (Engku, 2019). From the repayments, returns will be channelled to the equity-holders of the crowdfunding project through the crowdfunding platform as per the agreed term, whether annually, semi-annually or quarterly. In addition, some parts of the repayments will be channelled to waqf institutions as the charge for utilising the undeveloped waqf lands for the microfinance programme, and this will also cover a 1% management fee for Islamic microfinance institution for being an intermediary in managing the overall sources of fund and carrying out the waqf-based microfinance programme. Figure 2 illustrates the flow mentioned above and a description of the waqf and crowdfunding-based microfinance.\n\n\n5 Swot analysis\n\nThe proposed model addresses the issue of waqf properties, referring to crowdfunding to develop waqf properties and overcome the lack of financing problems to certain levels. Crowdfunding is an innovative tool that has been known for the past ten years, showing successful stories in the West and Muslim countries. However, there were a few successful platforms like MyWakaf.com.my, Ethiscrowd, Kapital boost, Launchgood, Global Sadaqah and others. By visiting these platforms, we have observed various successfully funded projects like housing projects, SME empowerment, waqf and other social campaigns.\n\nIn order to strengthen the position of the main argument of this paper, we would like to point out several articles written on the issue of undeveloped waqf properties. For example, Allah Pitchay et al. (2018) claimed that there are more than 11,000 thousand hectares of waqf lands in Malaysia, where only 7 percent of the total area has been developed due to a lack of financing. In addition, Dahlia Ibrahim and Haslindar Ibrahim (2013) emphasised the importance of Cash Waqf, which in our case will be in the form of obtaining a small amount of money invested from a large number of people in a Sharia-compliant manner through an online platform. This instrument is seen as a low-cost way to raise Sharia-compliant equity capital. Therefore, the proposed model, “Equity-based crowdfunding for waqf financing”, gives entrepreneurs access to a wide variety of capital, allowing them to mobilise money for financing.\n\nMoreover, the problem of locked financing could be unlocked using the crowdfunding platform, the funds raised from different donors via the internet. The collection of funds is to be realised quickly as the targeted audience is enormous. Biancone and Radwan (2019) assert that this is a unique model and could alleviate funding problems affecting the waqf industry.\n\nAlthough the proposed model appears at first glance to be effective and has excellent potential for success, this is a basic model, and further studies are needed to develop a comprehensive framework. Moreover, the framework must encompass different perspectives for effective implementation and execution. There is an actual need to address the regulatory framework governing crowdfunding platforms in Malaysia. In terms of compliance, the Equity-based crowdfunding for waqf financing structure needs to consider the necessary due diligence on the raised fund. There is a chance of money being collected from illegal sources or activities where investors might be involved in money laundering, drug trafficking, etc.\n\nSecondly, Waqf projects need millions of ringgits to be developed. Nevertheless, a platform like mywakaf.com was an initiative of AIBIM to crowdfund different Waqf projects. However, the platform failed to meet the target and raise funds for most projects. Hence, based on our study, the funds that can be raised through crowdfunding may be successful for small projects. However, this can be practical if the big project is divided into several campaigns launched on multiple platforms and with reasonable and attainable targets. Moreover, incorporating Islamic Finance products like Musharakah, Mudarabah and Sukuk will become necessary for the efficient and effective development of waqf projects.\n\nThirdly, this study is a conceptual paper with no empirical studies. Therefore, an empirical study must be considered for a robust finding and result. The following suggested areas be covered in the empirical studies:\n\n• Case study on the limitations of crowdfunding through the analysis of one of the existing platforms.\n\n• The acceptance of technology by potential entrepreneurs, a survey is needed to assess the acceptance level of investors.\n\n• Interview with the Awqaf administration and crowdfunding industry players to identify the possibilities of potential investments via the crowdfunding platform.\n\nISF’s concepts are based on the Islamic value of philanthropy, which includes standardised giving behaviours to meet the needs of the poor. This is in line with the United Nations’ belief that ISF assists governments and societies in meeting various development needs (Abdulkareem et al. 2021). Moreover, according to Khalil, Ab Rahman, Thaidi, and Rahman (2020), waqf could alleviate the community’s burden during and after the implementation of MCO in Malaysia. The benefits of waqf are beneficial not only to the community and society but also to the government and the nation’s development. Similarly, the proposed model in this paper aims to improve undeveloped waqf lands in Malaysia via collection of investment funds from global investors and multiple local and international shariah-compliant equity-based crowdfunding. The model will provide the waqf institution with adequate financing to implement a sustainable project like agriculture activities, which will provide job opportunities and generate income from the produced product to mitigate the sufferings of refugees and other vulnerable people during the pandemic.\n\nAnother structure in which the model may not succeed is the unskilful human capital Awqaf institutions have. For instance, the crowdfunding platform successfully funds a RM 20 million project. The fund will be channelled to the Awqaf institutions to manage the fund and the execution of the project. However, the project might not be fully implemented in the determined timeline if the human capital taking over the full responsibility for the project’s execution lacks expertise. There should also be a guarantee that the waqf institution managers will provide a periodic return to the investors. Moreover, lack of transparency is another issue that might arise if the crowd or investors are willing to track the progress of the funded project. In addition, a periodic report update of the project that will ultimately be shared with the crowd via the internet should be produced.\n\n\n6 Opportunities of implementing the wcm model in the eu\n\nMany European countries are directly affected by humanitarian crises, especially the Syrian refugee crisis due to the influx of legal and illegal migrants that creates issues including security, political and social cohesion. Most European countries prefer to shelter refugees in the Syrian borders in Turkey and prefer to provide a financial contribution to addressing the crisis (Parakkuth, 2017). One of the key causes of Britain’s decision to implement “Brexit” and alter the political landscape was the migrant issue (Siegfried, 2016). For improving the socio-economic conditions of these refugees settling in European countries, the WCM model can play an important role, as microfinance can offer sustainable and long-term solutions for the crisis of the refugees. Through learning and training, microfinance programmes may also assist refugees in enhancing their capabilities while still honouring their humanity and dignity (Parakkuth, 2017).\n\nIn recent years, Europe has seen growth in the microfinance industry. Given the effects of the current slow economic growth on underprivileged and disadvantaged communities and the need to ensure their social and financial inclusion, it still has significant growth potential. The role of microfinance is even more crucial, given the significant rise in self-employment and business formation. The recent growth of microfinance in Europe indicates that this industry is increasingly meeting the demands of immigrants and refugees who are still not eligible for formal financial services. Nearly 700,000 microloans, totaling more than 2 billion euros, were disbursed by various EU Microfinance Institutions (MFIs) in 2017 (Benaglio, 2019). These European MFIs can find the current WCM model useful in implementing their microfinance programmes. The benefits of a microfinance-based WCM model for refugees can be enormous in terms of both reducing poverty and minimising social exclusion, given the severely fragmented nature of the European economy and the lack of a widely acknowledged microfinance business model in this region.\n\n\n7 Conclusion\n\nDespite robust economic growth in the last decade, poverty continues to be a major problem in many parts of Asia-Pacific. In fact, poverty makes it difficult to pay for even the most basic necessities, such as food, decent housing, children’s healthcare, and clothing, to name a few. Moreover, poverty does not stand still. It can spread further if nothing is done to contain and eradicate it. In this context, microfinance can be considered an effective tool for eradicating poverty in different parts of the world, specifically in Malaysia. Microfinance is a critical component in developing sustainable livelihoods that are productive enough to provide disadvantaged households with a path out of poverty that is based on self-reliance and mutual aid.\n\nMoreover, the refugee phenomenon has been widely perceived as a temporary problem; however, the statistics of the UNHCR confirm that nearly two-thirds of refugees are usually stuck in protracted situations, helpless and hopeless. Using microfinance based on cash waqf and crowdfunding, these refugees can be offered the help they need to get out of the poverty trap and lead a decent life. Particularly for refugee microentrepreneurs, this microfinance programme can be immensely beneficial.\n\nAccording to the findings of this study, a large portion of waqf land remains idle. The Waqf institution is having trouble satisfying its cash requirements in order to develop specific lands (undeveloped lands). A lack of allocation is the most significant obstacle limiting their capacity to develop waqf land in Malaysia. As a result, the current study proposes a Waqf & Crowdfunding-based Microfinance concept to address the financial constraints of waqf institutions while enhancing the development of waqf land and contributing to SDGs 1 and 2 and national income. In addition, the model presented in this study can hugely benefit the distressed refugees and immigrants in Malaysia and the EU by effectively ameliorating their socio-economic conditions.\n\nThe main objective of this study was to develop a novel waqf model that can efficiently contribute towards poverty reduction in society, tailored explicitly for low-income households and refugees and immigrants to sustain financial independence. For this purpose, the necessary information was collected through the review of the existing scholarly literature. The major limitation of this study was that no primary data was collected from the experts in the waqf arena. Their opinions could have bolstered the arguments presented in this study about the usefulness of the model presented here.\n\nIt is expected that if the necessary steps are taken to implement the model presented here by the waqf institutions, it can play a significant role in alleviating the poverty of the refugee and immigrant people. Eventually, this will contribute towards reducing the suffering of these people and bringing smiles to their faces. In addition, future researchers working in this waqf area will have some new ideas to work on and improve the model to make it more effective and robust.\n\n\nAuthor’s contribution\n\nThe entire paper is written by the author.", "appendix": "Data availability statement\n\nNo data are associated with this article\n\n\nReferences\n\nAbdulkareem IA, Mahmud MS, Elaigwu M, et al.: Mitigating the Effect of Covid-19 on the Society Through the Islamic Social Finance. The Journal of Management Theory and Practice (JMTP). 2021; 2(1): 57–61. Publisher Full Text\n\nAddae-korankye A: Causes of Poverty in Africa: A Review of Literature. American International Journal of Social Science. 2014.\n\nAdministration of Islamic Religious Affairs (Terengganu) Enactment: Enactment No. 2 of 2001.2001. Reference Source\n\nAllah Pitchay A, et al.: Cooperative-Waqf Model: A Proposal to Develop Idle Waqf Lands in Malaysia. ISRA International Journal of Islamic Finance. 2018; 10(2): 225–236. Publisher Full Text\n\nBanerjee A, Duflo E: Poor Economics: A Radical Rethinking of the Way to Fight Global Poverty. Public Affairs. 2011. Reference SourceReference Source\n\nBenaglio N: The untapped potential of microfinance in Europe.2019. Reference Source\n\nBiancone PP, Radwan M: Social Finance and Financing Social Enterprises: An Islamic Finance Prospective. European Journal of Islamic Finance. 2019.\n\nChoo Hong L: Crowdfunding: Issues Pertaining to Financial Reporting and Assurance in Malaysia. Journal of Wealth Management & Financial Planning. 2018; 5(June): 17–24.\n\nIbrahim D, Ibrahim H: Revival of Waqf Properties in Malaysia. The 5th Islamic Economics System Conference (iECONS 2013) (September): 4–5. 2013.\n\nDi Pietro F: Equity Crowdfunding. Crowdfunding for Entrepreneurs; 2020. Publisher Full Text\n\nEngku RA: Successful models of social finance initiatives: Lessons from Amanah Ikhtiar Malaysia (AIM). 14th International Shari’ah Scholars Forum [ISSF 2019]. 2019.\n\nGlobal Monitoring Report: Global Monitoring Report 2009.2009. Publisher Full Text\n\nHaque T, Siwar C, Bhuiyan AB, et al.: Contributions of Amanah Ikhtiar Malaysia (AIM) microfinance to economic empowerment (EE) of women borrowers in Malaysia. Econ. Soc. 2019; 12(4): 21–42. Publisher Full Text\n\nHtay SNN, Sadzali NS, Amin H: An analysis of the viability of micro health takaful in Malaysia. Qualitative Research in Financial Markets. 2015; 7(1): 37–71. Publisher Full Text\n\nIbrahim SA, Roslen SNM, Salleh NM, et al.: Islamic Micro Financing: Crowdfunding as a Drive to Improve Financial Inclusion in Malaysia. International Journal of Engineering & Technology. 2018.\n\nInternational Monetary Fund: Poverty and distributional impacts of COVID-19: Potential channels of impact and mitigating policies.2020, April 16. Reference Source\n\nIsmail CZ, Salim NJ, Hanafiah NJA: Administration and management of waqf land in Malaysia: Issues and solutions. Mediterr. J. Soc. Sci. 2015; 6(4S2): 613–620. Publisher Full Text\n\nKhalil MAHM, Ab Rahman MF, Thaidi HAA, et al.: Advantages of temporary waqf in combating COVID-19 pandemic in Malaysia. INSLA E-Proceedings. 2020; 3(1): 167–180.\n\nKhanam D, Mohiuddin M, Hoque A: Financing micro-entrepreneurs for poverty alleviation: a performance analysis of microfinance services offered by BRAC, ASA, and Proshika from Bangladesh. J. Glob. Entrep. Res. 2018; 8(27). Publisher Full Text\n\nMahmood RH, Mustaffha N, Musafar Hameed LB, et al.: Pengurusan wakaf di Malaysia: Isu dan babaran. Proceedings 4th International Conference on Management and Muamalah 2017 (ICoMM). 2017. e-ISBN: 978-967-0850-XX-X.\n\nMarwan S, Haneef MA: Does doing good pay off? Social impact bonds and lessons for Islamic finance to serve real economy. Islam. Econ. Stud. 2019; 27(1): 23–37. Publisher Full Text\n\nMd Saad N: Microfinance and Prospect for Islamic Microfinance Products: The Case of Amanah Ikhtiar Malaysia. Advances in Asian Social Science. 2012.\n\nMohamad IAJ, Sofri Y, Pitchay AA: The contemporary model of waqf structure. The International Conference on Islamic Leadership and Management. 2016; 2016. Publisher Full Text\n\nMohamad ZS, Crendy TYT, Aniq Luqman SA, et al.: Refugees Life in Malaysia: A Review Paper. Malaysian Journal of Social Sciences and Humanities (MJSSH). 2021; 6(9): 437–446. Publisher Full Text\n\nMohammad TSM: Alternative development financing instrument for waqf properties. Malaysian Journal of Real Estate. 2009; 4(2).\n\nMohd AMH, Anuar A, Siti MM: Systematic literature review of waqf land development in Malaysia. International Journal of Advanced Research in Economics and Finance. 2020; 2(2): 70–78. e-ISSN: 2682-812X.\n\nMohd Ali N, Ahmad R, Shahnaz Mahdzan N, et al.: The need of an effective business model for waqf land development in Malaysia Financial Wellbeing of Middle income Malaysians.2015. Reference Source\n\nMohr J: SDGs as a Network of Targets. Kumu; 2016.\n\nNazrie Mohd Nor A, Kumar TS: The Chronology of Microfinance Development in Malaysia: A Review. KnE Social Sciences. 2019. Publisher Full Text\n\nNazrul HN, Siti NH, Salina K: Developing a comprehensive performance measurement system for waqf institutions. Int. J. Soc. Econ. 2017; 44(7): 921–936. Publisher Full Text\n\nNehme M: Regulating crowd equity funding - The why and the how. J Law Soc. 2018; 45: 116–135. Publisher Full Text\n\nNura AG, Asmak AR: The role of Islamic finance in the economic impact of covid-19 towards the attainment of maqasid al-Shariah: A case study of waqf institutions in Kano State, Nigeria. The Journal of Muamalat and Islamic Finance Research. 2020; 17(Special Issue). e-ISSN: 0126-595.\n\nOlalekan Yusuf T, Hakeem Mobolaji AI: The Role of Islamic Micro Insurance in Economic Growth and Development: The Nigerian Experience: A Case Study of Al-Barakah Microfinance Bank, Lagos. International Journal of Business and Commerce. 2012; 1(110): 106–122.\n\nParakkuth SAC: Economic Empowerment through Islamic Microfinance: Application to Refugee Settings. Hamad Bin Khalifa University; 2017. Doctoral dissertation.\n\nRapi MZH, Salaudeen AO, Ravi MIMR, et al.: Micro takaful programs for the poor: the Malaysian experience. Int. J. Account. 2022; 7(45): 65–81.\n\nSaad AA, Rehan R, Abukakar Usman A, et al.: The metaverse and Islamic financial contracts: The case of Ijarah. F1000Res. 2023; 12: 837. Publisher Full Text\n\nSadeq AM: Poverty alleviation: An Islamic perspective. Humanomics. 1997; 13(3/4): 110–134. Publisher Full Text\n\nSaiti B, Dembele A, Bulut M: The global cash waqf: a tool against poverty in Muslim countries. Qualitative Research in Financial Markets. 2020; 13: 277–294. 1755-4179. Publisher Full Text\n\nSanti M, Vika AQ, Ermita Y, et al.: The role of cash waqf as a source of micro business financing for strengthening the local economy: A case study in Gunung Kawi District, Malang Regency. Review of Integrative Business and Economics Research. 2021; 10(Supplementary Issue 1).\n\nSalaudeen AO, Zakariyah H: Challenges Hindering Islamic Microfinance Banks’ Sustainable Financial Inclusion: A Case of Al-Hayat Microfinance Bank in Ogun State, Nigeria. El-Barka: Journal of Islamic Economics and Business. 2022; 5(1): 24–50. Publisher Full Text\n\nSalaudeen AO, Sa’ad AA, Suleiman H: Sustainability and Operational Challenges of Islamic Microfinance in Nigeria: The Case of Al-Hayat Microfinance Bank. International Conference on Business and Technology. Cham: Springer International Publishing; 2022; pp. 457–473.\n\nSiti SA, Halim MN, Sharifzie MS, et al.: Analysis of corporate waqf model in Malaysia: An instrument towards muslim’s economic development. Indian Journal of Applied Business and Economic Research. 2016; 14(5): 2931–2944.\n\nSeibel HD: History matters in microfinance. Small Enterprise Development; 2003.\n\nSiegfried K: What does Brexit mean for refugees?.2016. Reference Source\n\nSrinivasan R, Sriram MS: Microfinance: An Introduction. IIMB Management Review; 2003.\n\nUK Government: Guidance: social impact bonds.2012. Reference Source\n\nUNHCR: Global Trends: Forced Displacement in 2018.2019. Reference Source\n\nWahab AA: Rethinking refugees as economically isolated: The Rohingyas participation in informal economy in Klang Valley, Malaysia. Journal of ASEAN Studies. 2017; 5(2): 100–118.\n\nWorld Bank: World Development Report 2020 — Global Value Chains: Trading for Development. World Bank Group; 2019.\n\nWorld Bank: COVID-19 to add as many as 150 million extreme poor by 2021. World Bank Blog.; 2020, October 13th. Reference Source\n\nWorld Bank: Updated estimates of the impact of COVID-19 on global poverty: Looking back at 2020 and the outlook for 2021. World Bank Blog; 2021. Reference Source\n\nWorld Bank Group: COVID-19 to add as many as 150 million extreme poor by 2021.2020, October 13th. Reference Source\n\nZakariyah H, Salaudeen AO, Othman AHA, et al.: Enhancing waqf management through fintech in Malaysia. Journal of Emerging Economies and Islamic Research. 2022; 10(2): 62–73. Publisher Full Text\n\nZakariyah H, Salaudeen AO, Othman AHA, et al.: The determinants of financial technology adoption amongst Malaysian waqf institutions. Int. J. Soc. Econ. 2023; 50(9): 1302–1322. Publisher Full Text" }	[ { "id": "305945", "date": "02 Aug 2024", "name": "Sherin Kunhibava", "expertise": [ "Reviewer Expertise Islamic finance 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\n1. Introduction has to explain the objective of the study clearly, and include some information on Malaysia. 2. Methodology should explain why Malaysia is chosen as the jurisdiction of study. 3. minimum wage in Malaysia is RM1500. 4. At page 5 the author states ‘The relevance behind charging on utilisation of the UWL instead of giving it away’ however waqf land cannot be given away as it belongs to Allah swt. Please rectify this whole part. 5. At page 7 – it is stated ‘The provisions mentioned in Section 63 of the Administration of Islamic Religious Affairs (Terengganu) Enactment, No. 2 of 2001’. This is the regulation for only one SIRC, each state in Malaysia has their own enactment. So this has to be mentioned. 6. The role of the recipients (refugees) have to be added to the flow. Including the challenges, unskilled workers, location of waqf lands, etc. 7. There should be included the role and support of the Ministry of Agriculture and Food Security.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "301926", "date": "03 Sep 2024", "name": "Sebastian Herman", "expertise": [ "Reviewer Expertise Islamic social finance and economics thought" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 very interested, the model could be acceptable, it is more appropriate when the author uses ANP to test the model. This research aims to develop an effective Waqf model to reduce poverty among refugees in Malaysia. By integrating Islamic principles, the model is expected to create sustainable resources that support education, healthcare, and micro-enterprises for refugees. The study will also explore collaborations with Islamic financial institutions and NGOs, and address legal and sustainability challenges to ensure long-term impact.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-763
https://f1000research.com/articles/13-126/v1	20 Feb 24	{ "type": "Review", "title": "Life of Pi: Exploring functions of Pi16+ fibroblasts", "authors": [ "Erika E. McCartney", "Yein Chung", "Matthew B. Buechler", "Erika E. McCartney", "Yein Chung" ], "abstract": "Fibroblasts are mesenchymal cells that are responsible for creating and maintaining tissue architecture through the production of extracellular matrix. These cells also play critical roles in processes such as wound repair and immune modulation in normal tissues and various disease states including fibrosis, autoimmunity, and cancer. Fibroblasts have a complex repertoire of functions that vary by organ, inflammatory state, and the developmental stage of an organism. How fibroblasts manage so many functions in such a context-dependent manner represents a gap in our understanding of these cells. One possibility is that a tissue-resident precursor cell state exists that provides the fibroblast lineage with flexibility during growth, inflammation, or other contexts that require dynamic tissue changes. Recent work has suggested that a precursor fibroblast cell state is marked by expression of Peptidase inhibitor 16 (Pi16). This review aims to concatenate and compare studies on fibroblasts that express Pi16 to clarify the roles of this cell state in fibroblast lineage development and other functions.", "keywords": [ "fibroblast", "Pi16" ], "content": "1. Introduction – On site fibroblast precursors\n\nCells of the fibroblast lineage are found across the entire body, where they maintain tissue architecture through the production of extracellular matrix, interact with immune cells, and contribute to a wide range of pathologies such as fibrosis, cancer, and autoimmunity.1 Understanding how this functional heterogeneity occurs and its role in immune homeostasis and inflammation has been a challenge. Parabiosis experiments suggest that fibroblasts that arise during inflammation are seeded from local, in situ local precursors, indicating that a circulating progenitor cell is not a likely source of new fibroblasts in tissue.2,3 One means by which fibroblasts may achieve parallel generalized function such as ECM remodeling, while also having context-specific phenotypes is via a precursor cell that resides within tissues. In this conceptual model, a fibroblast cell state exists that can develop into more specialized fibroblast subtypes based on context specific signalling, providing this essential cell lineage flexibility during development and disease conditions.\n\nIn 2021, Turley and colleagues used a combination of 28 publicly available mouse fibroblast single cell RNA sequencing (scRNAseq) datasets to create a fibroblast transcriptional atlas to address this question. They identified that cells of the fibroblast lineage could be divided into either context-specific clusters that were influenced by tissue (e.g., Ccl19+ fibroblastic reticular cells in secondary lymphoid organs) or clusters that were found in all tissues (lymph node, omentum, thymus, lung, skin [ear and flank], adipose [epidydimal, subcutaneous, and brown], pancreas, and heart).4 These conserved clusters expressed Dermatopontin (Dpt) and could be further characterized by their expression of Peptidase inhibitor 16 (Pi16) and Collagen 15a1 (Col15a1). The existence of these subsets in the aforementioned tissues was validated in vivo using a Dpt.IRES.cre.ERT2;Rosa26.Lox-Stop-Lox.YFP (iDptYFP) reporter mouse.4\n\nThe role of cell states in biology can be challenging to unequivocally demonstrate as laboratories use different techniques, employ distinct nomenclatures, and examine different tissues or contexts. Cells that transcriptionally match Pi16+ fibroblasts have been termed universal fibroblasts,4 adventitial fibroblasts,5 interstitial progenitor cells,6 multipotent progenitor cells,7 fibro-adipogenic progenitors,8 and fibro-inflammatory progenitors.9,10 In silico analyses have revealed a list of marker genes that are expressed by Pi16+ fibroblasts, including Dpp4 (Dipeptidyl peptidase-4), Cd55, Ackr3 (atypical chemokine receptor, encodes CXCR7), Anxa3 (Annexin A3), and Il33 (See Table 1).\n\nThe presence of transcriptionally analogous fibroblast states across the body may suggest a Pi16+ fibroblast niche that exists across tissues. Pi16+ fibroblasts have been identified near vasculature numerous tissues including the lung,4 tonsil,11 muscle,8,12 and mammary gland,13 and near neural structures in the muscle12 and central nervous system.14 Notably, fibroblasts with a Pi16+ phenotype were also found away from blood vessels, instead associated with the reticular interstitium,15 a band of fluid-filled space enmeshed with fibrous collagen found within and across tissues including skin and subcutaneous adipose.6,16 These shared niches may either suggest a conserved function across tissues or may be the driving force in maintaining transcriptional similarity across tissues.\n\n\n2. Lineage functions of the Pi16+ fibroblast\n\nOur understanding of fibroblast biology has expanded greatly since the development of scRNAseq. This approach uncovered distinct functional fibroblast subsets within a single tissue. It was in silico trajectory analysis,4 since performed by numerous groups,14,16,17 that first suggested a lineage relationship in which Pi16-expressing cells give rise to the various tissue specific fibroblast subsets, possibly through a Col15a1+ intermediate cell state. It is worth noting that in several instances, the Pi16+ fibroblast cluster was set as the initial root cluster during lineage inference.4,13,16,17\n\nEmpirical validation of in silico approaches that suggest a lineage relationship amongst fibroblasts beginning at the Pi16+ stage has been examined within adipose tissue. Adipogenesis is the process by which adipocytes, key cells for energy storage, develop. This can occur via two processes: hyperplasia, the development of adipocytes from a precursor cell, and hypertrophy, the process by which adipocytes increase in size to accommodate increased lipid storage. The contribution of fibroblasts to adipogenesis via hyperplasia was first confirmed using genetic tools in vivo in 2013 when adipocytes in white adipose tissue were shown to be derived from Platelet-derived growth factor alpha (PDGFRa)+ cells using a Pdgfra.cre mouse.18 In vivo adipogenesis is complex and may exhibit temporal contours.19,20 Current models suggest early adipogenesis is dependent on Peroxisome proliferator-activated receptor gamma (Pparg) in PDGFRA-expressing cells.21 However, in later-stage adipogenesis, non-fibroblastic mural cells may contribute to the adipocyte pool.20\n\nA study by Merrick et al., described two fibroblast populations within white adipose tissue that contribute to adipogenesis in a stepwise fashion. One population was defined by Pi16, Cd34, and CD26 (encoded by Dpp4), while the other, a preadipocyte was marked by ICAM1 and shares attributes with Dpt+Col15a1+ population identified by Buechler and Pradhan et al.4,22 In in vitro adipogenesis assays, both populations of fibroblasts exhibited adipogenic capacity. The ICAM1+ preadipocytes gave rise to adipocytes faster and in greater numbers than the Pi16+ populations. This discrepancy in kinetics was attributed to the preadipocytes being a more differentiated cell state.22 To assess their in vivo contributions to adipogenesis, Pi16+ and ICAM1+ cells isolated from congenically-marked (TdTomato) mice were transplanted into adipose depots of recipient mice. Transplantation of Pi16+-like cells resulted in TdTomato+ ICAM+ preadipocytes as well as mature adipocytes, while transplant of ICAM1+ cells resulted only in mature adipocytes.22 This elegant cell transfer system confirmed that Pi16+ cells can as a precursor for the adipocyte lineage and that differentiation occurs in a stepwise manner from through an ICAM1+ (Col15a1+) intermediate in vivo.\n\nYet, these results did not confirm the in situ role of Pi16+ cells in vivo within adipose tissue. To this end, the same group used a Dpp4.creERT2;GFP mouse line, to reveal that CD26+ cells contributed to mature adipocytes when mice were on both a normal chow or high fat diet (HFD).23 Mice on HFD also exhibited a greater number of GFP+ adipocytes after 24 weeks. This mouse model also confirmed the finding that CD26+ cells give rise to ICAM1+/Col15a1+ preadipocytes in vivo.\n\nContexts outside the adipogenesis field also indicate that Pi16+ fibroblasts may function as a precursor cell for the fibroblast lineage. The Underhill and Rossi groups proposed that Hypermethylated in cancer 1 (Hic1) marked tissue resident mesenchymal progenitors. The transcriptional definition of Hic1+ cells, Pdgfra, Thy1, and Gli1, suggests these cells may represent Pi16+ cells in the muscle. Pulse-chase experiments in the heart using a Hic1creERT2;Rosa26TdTomato mouse, which specifically labels Hic1+ fibroblasts with TdTomato, demonstrated that a small proportion of specialized cardiac fibroblasts were TdTomato+ at both 7 and 35 days post-pulse.24 This suggests that fibroblasts which share phenotypic characteristics with Pi16+ fibroblasts contribute to specialized fibroblast subsets at steady state in the heart.\n\nInterestingly, Hic1 may also functionally regulate fibroblast biology in vivo. Cells that exhibit the phenotype of Pi16+ fibroblasts were shown to be in a state of quiescence in the steady state, with minimal proliferation in uninjured tissues.8 The ubiquitous deletion of Hic1 using UBC.cre.ERT2;Hic1flox/flox mice, led to the rapid expansion of fibroblasts, quantified by flow cytometry and the incorporation of EdU (5-ethynyl-20-deoxyuridine) in the Pi16+-like population.8 The deletion of Hic1 also had an activating effect on these cells, comparison of differentially expressed genes showed a substantial overlap of genes with injury-activated Hic1+ cells.8 The deletion of Hic1 in Pdgfra+ fibroblasts (Pdgfra.CT2;Hic1Flox/Flox mouse) resulted in epicardium fibrosis and accumulation of adipocytes that drastically reduced cardiac function.24 This study not only suggests Hic1 is required for the quiescence of Pi16+-like fibroblasts but demonstrates the multipotent capacity of these cells.\n\nThe quiescence of Pi16+ cells, which can be unlocked by loss of Hic1 using genetic tools,24 may have physiological relevance. In the spared nerve injury (SNI) model of chronic neuropathic pain, Pi16 expression was increased in both the sciatic nerve and lumbar dorsal root ganglia 8 days post-injury compared to sham surgery controls. An increased number of Pi16+ fibroblasts were also demonstrated in proximity to the injury site.14 This suggests that while Pi16+ fibroblasts are mitotically inactive in healthy tissues, expansion may be induced by damage to facilitate tissue repair.\n\nBuechler and Pradhan et al., observed Pi16+ fibroblasts across diseased tissues in mice and humans. Transcriptionally, Pi16+ fibroblasts retained expression of genes associated with stemness during inflammation and trajectory analysis maintained the lineage relationship from Pi16+ to Col15a1+ before differentiation into specialized tissue-specific fibroblasts or pathological Lrrc15+ myofibroblasts.4 In silico trajectory analysis of fibroblast clusters within a dorsal skin wound validated this concept in the skin, showing that that Pi16+ fibroblasts within the fascia adopt a proinflammatory state before differentiating into myofibroblasts.16 The differentiation of fibroblasts with a Pi16+ phenotype to myofibroblasts in wound repair was shown to be required as blocking this differentiation resulted in weakened and poorly organized ECM fibers, and poor wound closure.16\n\nLineage tracing has confirmed that in situ fibroblasts may act as precursor cells to more specialized fibroblasts. Using the iDptYFP mouse, Turley and colleagues showed in two studies that in subcutaneous and orthotopic models of pancreatic cancer LRRC15+ myofibroblasts derive from Dpt+ fibroblasts.4,25 A study by Houthuijzen et al. found that different subsets of cancer associated fibroblasts (CAFs) in breast cancer are derived from CD26+ and CD26- fibroblasts within the mammary gland.26 CD26- fibroblasts gave rise to myofibroblastic CAFs, while CD26+ cells preferentially produced inflammatory CAFs. Despite this, CD26+ CAFs also showed features in line with myofibroblastic CAFs, suggesting that either inflammatory CAFs are functionally plastic and can contribute to both inflammatory processes and myofibroblastic functions or myofibroblasts may develop directly from a CD26+ intermediate. Lineage tracing experiments using a tool such as the Dpp4.creERT2;GFP mouseline23 would be helpful to confirm the contribution of CD26+Pi16+ cells to these CAF phenotypes.\n\nThe contribution of Pi16+ cells to specialized fibroblasts and adipocytes has been demonstrated in various tissues at steady state as well as in numerous disease contexts and injury models, indicating a progenitor role for context-specific fibroblast subsets. The ability of these cells to generate numerous cell states within the same tissue, as seen upon Hic1 deletion in the heart24 and by contribution to distinct CAF subsets in breast cancer,26 affirms the multipotent capacities of these cells in vivo.\n\n\n3. Non-lineage functions of the Pi16+ fibroblast\n\nThe concept of a fibroblastic stem-like cell marked by Pi16 that resides across tissues to give rise to other fibroblast cell types is simple and conceptually attractive. Yet, if Pi16 marks a fibroblast lineage, a concept that has not yet been proven, the function of these cells in vivo may be distinct from a strictly precursor role. Alternatively, these cells may serve several functions, either due to inherent plasticity, heterogeneity within Pi16+ cells, temporal regulation during development, or other factors. Indeed, some studies have suggested a non-precursor role for Pi16+ cells.\n\nArostegui et al. described an embryonic population of mesenchymal progenitor cells marked by Hic1 in the limb bud that persist into adulthood. These cells were shown to overlap transcriptionally with Pi16+ fibroblasts.12 In this study, Hic1+ cells appeared in the limb bud at embryonic day 11.5 (E11.5) and these cells co-localized with CD31+ endothelial cells, consistent with other studies.4,12,14 ScRNAseq of Hic1-labeled cells from E11.5-E16.5 suggested that these cells operate as a precursor population in the developing limb bud. Cells from earlier time points (E11.5 and E12.5) clustered together and were transcriptionally distinct from Hic1-labeled cells at E14.5 and E16.5. The E11.5+12.5 cluster expressed genes associated with a progenitor phenotype, while E14.5+E16.5 cells formed clusters suggestive of several distinct differentiated phenotypes, including numerous fibroblast subsets, chondrocytes, and pericytes.12 Trajectory analysis of this data set suggests a direct lineage relationship between Hic1+ cells and each of the differentiated phenotypes identified at E14.5 and E16.5. A pulse of tamoxifen at E10, labelling cells currently expressing Hic1+ with TdTomato, allowed for in vivo validation of this finding. By E18.5, Hic1+ cells contributed to chondrocytes, tenocytes, pericytes, and numerous fibroblast subsets, as visualized by immunofluorescence.12 This indicates that Hic1+ cells are a functional and multipotent progenitor of numerous mesenchymal cell types during embryonic limb development. Interestingly, within the fibroblasts marked by the Hic1 allele, a bifurcation existed at E18.5 between Pi16+ cells (Fibroblast I) and other fibroblasts (Fibroblast II), suggesting that Pi16+ cells may represent a distinct, non-precursor lineage. Consistent with this finding, in developing lymph nodes, Pi16+ fibroblasts were identified in silico but were suggested to not operate as a stem-like reservoir population.27 However, the contours of Pi16+ fibroblast development, and that of the entire fibroblast lineage, across tissues and timepoints during embryogenesis remains largely unexplored.\n\nIn the context of skin, Rinkevich et al. described a population of cells marked by the gene Engrailed-1 (En1). The En1.cre allele first emerges in the skin at embryonic day 16.5 (E16.5).28 The cells, named En1 positive fibroblasts (EPFs), become the dominant fibroblast population by P30. Similar to Pi16+ cells as described by Buechler and Prahdan et al. the EPFs were marked by CD26 (Dpp4) expression and were the dominant fibroblast type in the adult skin.4,28 EPFs directly contributed to skin architecture maintenance and wound repair through the production of collagen. In a later study by Rinkevich et al. EPFs within the skin fascia were described as expressing both Dpt and Pi16.16 Interestingly, the inhibition of CD26 in the skin led to delayed wound healing and decreased scar size,28 which may suggest a crucial role of CD26 in the activation and/or function of Pi16+ cells.\n\nPi16+ fibroblasts may also be pro-fibrotic; Gupta and colleagues identified fibro-inflammatory progenitors (FIPs) using a PdgfrbrtTA;TRE-cre;Rosa26RmT/mG “MuralChaser” mouse in both visceral and subcutaneous adipose depots by scRNAseq.29 FIPs were shown to exhibit the highest expression of extracellular matrix transcripts suggestive of a pro-fibrotic function.29,30 FIPs also exhibit some potential to develop into adipocytes, suggesting dual roles as progenitors and key players in fibrosis, possibly due to heterogeneity within this population.9 Analysis in the murine lung has supported the concept that some Pi16+ cells may be stem-like whereas other Pi16+ cells adopt roles during fibrosis.31\n\nPi16 expressing fibroblasts have been identified in numerous secondary lymphoid organs across species. These cells have similar transcriptional profiles across tissues but exhibit some tissue level imprinting as defined by scRNAseq. Cell-cell interactions were predicted using in silico approaches and suggested Pi16+ cells produced a variety of chemokines and cytokines which likely have a pro-inflammatory effect on both myeloid cells and lymphocytes.11,32 Ludewig and colleagues identified PI16+ fibroblasts localized to vessel rich regions of the human tonsil and make direct contact with B cells and T cells.11 In vitro assays confirmed that these tonsillar Pi16+ fibroblasts were potent mediators of T cell activation.11 In the muscle injury model, Hic1+ fibroblasts cells co-cultured with activated CD3+ T cells demonstrated that Pi16+ fibroblasts induce significant proliferation in both CD4+ and CD8+ T cells. CD25, a marker of T cell activation, was also seen to increase in response to co-culture with Pi16+ cells in comparison to stimulation with PHA alone.14\n\nThe presence of Pi16+ fibroblasts in a shared niches across tissues may suggest a conserved function across the body. Pi16+ fibroblasts may mediate extravasation of cells into tissues.12,14 Their direct role in this process is not understood but may be through enzymatic activity of Pi16 itself or CD26, which are both known to modify proteins associated with immune cell trafficking.33,34 Pi16 knockout mice showed a protection from pain response after SNI, decreased leukocyte infiltration into the dorsal root ganglia, as well as decreased endothelial permeability.14 Depletion of fibroblastic progenitor cells in the muscle showed a similar decrease in immune cell accumulation post-injury.8 This suggests Pi16+ cells play a role in immune infiltration upon injury, which is consistent with their proximity to vessels across tissues and the enzymatic functions enzymatic activity of Pi16 and CD26, which are both known to modify proteins associated with immune cell trafficking.33,34 If Pi16+ fibroblasts are required for extravasation and immune infiltration, therapeutic exploitation of these pathways in the tumour microenvironment to modify cold tumours and immune deserts into immune rich tumors.1,8\n\n\nConclusions\n\nPi16+ fibroblasts have been found across tissues with similar gene expression profiles and localization patterns through a combination of in vivo and in silico approaches. In some contexts, Pi16+ fibroblast-like cells have exhibited the potential to act as a precursor cell for fibroblasts and fibroblast-derived cells, including but not limited to myofibroblasts, adipocytes, and CAFs (Figure 1). These cells are seen across the body at both steady state and in many disease models, suggesting a functional importance in all contexts. Further characterization of these cells across tissues and contexts, however, is important to confirm each of these studies is truly describing the same cell state. The epigenetic landscape of these cells has also not been described, which may provide important insight into the progenitor capacity of Pi16+ fibroblasts. However, until comprehensive lineage tracing experiments using genetic tools is be conducted, the concept of a step-wise lineage relationship in which all fibroblasts across all tissues develop from Pi16+CD26+ cells cannot be confirmed.\n\nPi16 expressing fibroblasts, driven by TGF-β and sheer stress, contribute to the production of specialized fibroblasts at homeostasis and myofibroblasts in inflammation and injury. They also may contribute to the production of extracellular matrix and the recruitment and activation of immune cells through the production of inflammatory cytokines.\n\nIndeed, whether Pi16 marks a lineage of fibroblasts at all has not yet been shown – it is possible that Pi16 is a biomarker for fibroblasts that have seen TGF-β11 or experienced sheer-stress35 (Figure 1). Additionally, it is a possibility that Pi16+ and Co1l5a1+ fibroblasts act as independent progenitors to distinct fibroblast lineages, such as pro-fibrotic progenitors and adipocyte precursor cells as seen in adipose tissues.29,30 Further, cells within the fibroblast lineage may not become terminally differentiated states and are instead all phenotypically and functionally plastic given the correct cues.36,37 Indeed, in many of the studies examined, the two Dpt+ fibroblast subsets4 are the predominant and sometimes sole fibroblasts in the examined tissue and are therefore the only fibroblasts available to respond to injury.\n\nOutside of potential progenitor roles, Pi16+ fibroblasts have been implicated in immunity, either through the direct activation of lymphocytes in the tonsil or through infiltration of immune cells after injury, potentially through promotion of increased endothelial permeability. While Pi16+ cells appear to be localized near vasculature in many of the examined tissues, their effects on endothelial cells have not been thoroughly explored outside of the brain.\n\nAs Pi realizes in Yann Martel’s novel the Life of Pi, human existence harbors complexity and interdependency. The same sentiments may apply to our understanding of the fibroblast lineage and the cells within it. Fibroblasts are heterogeneous, allowing for their large number of functions throughout the body in tissue- and context-specific manners. The role of fibroblasts in a variety of pathologies such as cancer and autoimmunity is only recently being appreciated and understanding how fibroblasts develop and function and how these activities contribute to disease will be crucial in the development of effective therapeutics. Understanding how the Pi16+ fibroblast functions and its ability to act in a both stem-like and immunomodulating fashion is a key first step in this process.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nKoliaraki V, Prados A, Armaka M, et al.: The mesenchymal context in inflammation, immunity and cancer. Nat. Immunol. 2020; 21: 974–982. Publisher Full Text\n\nArina A, et al.: Tumor-associated fibroblasts predominantly come from local and not circulating precursors. Proc. Natl. Acad. Sci. 2016; 113: 7551–7556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBucala R, Ritchlin C, Winchester R, et al.: Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. J. Exp. Med. 1991; 173: 569–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuechler MB, et al.: Cross-tissue organization of the fibroblast lineage. Nature. 2021; 593: 575–579. PubMed Abstract \| Publisher Full Text\n\nDahlgren MW, et al.: Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches. Immunity. 2019; 50: 707–722.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerrick D, et al.: Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science. 2019; 364: eaav2501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKabat AM, et al.: Resident TH2 cells orchestrate adipose tissue remodeling at a site adjacent to infection. Sci. Immunol. 2022; 7: eadd3263. PubMed Abstract \| Publisher Full Text\n\nScott RW, Arostegui M, Schweitzer R, et al.: Hic1 Defines Quiescent Mesenchymal Progenitor Subpopulations with Distinct Functions and Fates in Skeletal Muscle Regeneration. Cell Stem Cell. 2019; 25: 797–813.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao M, et al.: Pathologic HIF1α signaling drives adipose progenitor dysfunction in obesity. Cell Stem Cell. 2021; 28: 685–701.e7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHepler C, et al.: Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. elife. 2018; 7: 771. Publisher Full Text\n\nMartin AD, et al.: PI16+ reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches. Nat. Immunol. 2023; 24: 1138–1148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArostegui M, Scott RW, Böse K, et al.: Cellular taxonomy of Hic1+ mesenchymal progenitor derivatives in the limb: from embryo to adult. Nat. Commun. 2022; 13: 4989. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshitake R, et al.: Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium. Front. Cell Dev. Biol. 2022; 10: 850568. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinghmar P, et al.: The fibroblast-derived protein PI16 controls neuropathic pain. Proc. Natl. Acad. Sci. 2020; 117: 5463–5471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenias PC, et al.: Structure and Distribution of an Unrecognized Interstitium in Human Tissues. Sci. Rep. 2018; 8: 4947. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRinkevich Y, et al.: CD201+ fascia progenitors choreograph injury repair.2022. Publisher Full Text\n\nKnights AJ, et al.: Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 in osteoarthritis. Ann. Rheum. Dis. 2023; 82: 272–282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerry R, Rodeheffer MS: Characterization of the adipocyte cellular lineage in vivo. Nat. Cell Biol. 2013; 15: 302–308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang Y, Berry DC, Tang W, et al.: Independent Stem Cell Lineages Regulate Adipose Organogenesis and Adipose Homeostasis. Cell Rep. 2014; 9: 1007–1022. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShin S, et al.: Dynamic control of adipose tissue development and adult tissue homeostasis by platelet-derived growth factor receptor alpha. elife. 2020; 9: e56189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosen ED, et al.: PPARγ Is Required for the Differentiation of Adipose Tissue in vivo and In Vitro. Mol. Cell. 1999; 4: 611–617. Publisher Full Text\n\nMerrick D, et al.: Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science. 2019; 364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStefkovich M, Traynor S, Cheng L, et al.: Dpp4+ interstitial progenitor cells contribute to basal and high fat diet-induced adipogenesis. Mol. Metab. 2021; 54: 101357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoliman H, et al.: Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors. Cell Stem Cell. 2020; 26: 205–220.e8. PubMed Abstract \| Publisher Full Text\n\nKrishnamurty AT, et al.: LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity. Nature. 2022; 611: 148–154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHouthuijzen JM, et al.: CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer. Nat. Commun. 2023; 14: 183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPezoldt J, et al.: Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes. Nat. Commun. 2018; 9: 3903–3914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRinkevich Y, et al.: Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science. 2015; 348: aaa2151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHepler C, et al.: Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. elife. 2018; 7: e39636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao M, et al.: Pathologic HIF1α signaling drives adipose progenitor dysfunction in obesity. Cell Stem Cell. 2021; 28: 685–701.e7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsukui T, et al.: Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis. Nat. Commun. 2020; 11: 1920. Publisher Full Text\n\nLütge M, et al.: Conserved stromal–immune cell circuits secure B cell homeostasis and function. Nat. Immunol. 2023; 24: 1149–1160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRegn M, et al.: Peptidase inhibitor 16 is a membrane-tethered regulator of chemerin processing in the myocardium. J. Mol. Cell. Cardiol. 2016; 99: 57–64. PubMed Abstract \| Publisher Full Text\n\nHu X, Wang X, Xue X: Therapeutic Perspectives of CD26 Inhibitors in Imune-Mediated Diseases. Molecules. 2022; 27: 4498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHazell GGJ, et al.: PI16 is a shear stress and inflammation-regulated inhibitor of MMP2. Sci. Rep. 2016; 6: 39553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalminen A: The plasticity of fibroblasts: A forgotten player in the aging process. Ageing Res. Rev. 2023; 89: 101995. PubMed Abstract \| Publisher Full Text\n\nFoster DS, et al.: Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin. Cancer Cell. 2022; 40: 1392–1406.e7. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "264918", "date": "13 May 2024", "name": "Natalia Pikor", "expertise": [ "Reviewer Expertise Stromal cell immunobiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBuechler and colleagues have prepared a comprehensive review of the many tissue settings and experimental models in which Pi16+ cells (or their transcriptionally similar counterparts) are demonstrated to respond to biological perturbation. There are minor comments that would be recommended to increase the clarity between lineage and non-lineage functions of Pi16+ fibroblasts.\n\nIn section 2., the authors describe experiments in which Pi16+ fibroblasts or their counterparts have been shown to serve as progenitors for more specialized fibroblastic cell types in vitro or using lineage-tracing models. Collectively the cited literature discusses the role for Pi16+ fibroblasts to give rise to adipocytes or to ECM-producing or contractile fibroblasts (in epicardial fibrosis or as myofibroblastic cancer associated fibroblasts in tumors). However, some of the studies quoted in the non-lineage functions of Pi16+ fibroblasts section appear to reflect a progenitor phenotype of Pi16+ or Pi16+-like cells. This includes the cited work by Rinkevich and colleagues, as well as pro-fibrotic function of fate-mapped 'mural chaser' cells in the adipose tissue. If one assumes that fibroblasts that give rise to CAFs with an ECM-enriched gene signature represent a progenitor-progeny relationship, is this not also the case for fibroblasts that differentiate to produce ECM and cover a scar in a tissue injury model?\n\nWhat appears to stand out are Pi16+ cells in lymphoid organs. Unlike the perivascular progenitors that receive Lymphotoxin signals to differentiate into fibroblastic reticular cells (Cheng HW et al., 2019 Nat. Comm. (Ref 1 )and Prados A. et al., 2022, Nat. Imm (Ref 2)), Pi16+ reticular cells do not appear to be FRC progenitors (Pezoldt et al. 2018 Nat. Comm) [Ref4]. Nevertheless, these cells are expanded in inflammation, and encode a stemness gene signature, as shown in chronically-inflamed tonsils (De Martin et al., 2023 Nat. Immunol. (Ref 3)). Perhaps, it is the case that they are either more inflammatory, as the authors have discussed, or respond according to the nature of the tissue injury.\n\nConsidering the above mentioned points, the authors may consider to reorganize these sections, and order the sections reflecting the graphical representation in Figure 1.\n\nFigure 1 may be clarified. Fibroblasts activated by inflammation or injury may be those that recruit or activate immune cells (as in lymphoid organs) or exhibit an ECM-depositing phenotype (wounding or CAFs), respectively.\n\nMinor comments:\n\nFigure 1: It would be helpful to cite the relevant references related to the 4 feature of Pi16+ function.\n\nThe authors may consider discussing mural cells as fibroblasts. At present there is a discrepancy between non-fibroblastic mural cells – relating to reference 20 and adipocyte progenitors and Pi16+ fibroblasts derived from Pdgfrb+ mural cells – relating to reference 29. Are vascular smooth muscle cells and pericytes fundamentally not a fibroblastic cell type? After all, adipocytes are included as specialized fibroblasts.\n\nReference 16 is mis-cited.\n\nIn reference 27 – it is not clear which CD34+ exhibits the Pi16+ phenotype\n\nIt is likely that the reticulated interstitium, like the fascia, is highly vascularized. Thus, although not mural cells, Pi16+ cells in these tissues may also be considered in proximity to the vasculature.\n\nSmall grammatical typos:\n\nSection 1, Paragraph 1 of the introduction: 'fibroblasts that arise during inflammation are seeded from local, in situ local precursors …'\n\nSection 1, Paragraph 4: 'Pi16+ fibroblasts have been identified near the vasculature in numerous tissues...'\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": "286893", "date": "24 Jun 2024", "name": "Dongsheng Jiang", "expertise": [ "Reviewer Expertise Fibroblast heterogeneity", "Scarring and Fibrosis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBuechler and colleagues have summarized two types of functional roles of Pi16+ cells through extensive scRNA-seq analysis, alongside in vitro and in vivo models in both physiological and pathological conditions. Below are some minor comments to help provide a more comprehensive profile of Pi16+ fibroblasts.\nIn the introduction, the authors introduce the concept that a precursor fibroblast state exists, which develops into specialized fibroblast subsets or states based on context-specific signalling. Following this, the authors give a comprehensive summary of Pi16+ fibroblast subsets from different tissues, using various animal and disease models. The table 1 is very informative.\nIn the subsequent lineage function section, the authors describe the precursor phenotypes of Pi16+ fibroblasts, and attempt to demonstrate that Pi16 is a marker that identifies the precursor fibroblast state previously introduced. Furthermore, by highlighting Phenotypic similarities after targeted cell depletion, the authors provide a link between Pi16+ and Hic1+ fibroblasts, and even with CD201+ or CD26 (Dpp4)+ fibroblasts. However to substantiate the equivalency of Hic1+ fibroblasts (or CD201+ or CD26+ fibroblasts) to Pi16+ fibroblasts, more evidence is needed. This could include coexpression at the mRNA level from sequencing data or coexpression at the protein level shown by, for example, immunofluorescence. Reinforcing this evidence would strengthen the conceptual claim.\nIn the next section, the authors elucidate the non-precursor roles of Pi16+ fibroblasts, particularly their functions in inflammation, fibrosis and scarring. On Page 5, in the last paragraph the following statements regarding EPFs are not fully correct: “The En1.cre allele first emerges in the skin at embryonic day 16.5 (E16.5). The cells, named En1 positive fibroblasts (EPFs), become the dominant fibroblast population by P30.” Please refer to the follow-up study described in Jiang et al., 2018, Nat Cell Biol, to revise these two sentences. Furthermore, the study by Mascharak et al., 2021, Science, is also worth mentioning. It is essential to point out that En1 is a lineage marker of fibroblasts. It is expressed only during a very short time window in early embryonic development (Jiang et al., 2018 [Ref -1]), or reactivated upon injury (Mascharak et al., 2021[Ref-2]). Additionally, the cited Reference 16 is a BioRxiv paper that has since been published in Nature (Correa-Gallegos et al., 2023 [Ref -3]). Please update this reference.\nSmall grammatical errors: verbs are missing in the following three sentences.\n1. On page 4, Paragraph 3: “This elegant cell transfer system confirmed that Pi16+ cells can...”\n2. On page 4, Paragraph 6: “The deletion of Hic1 also had an activating effect on these cells, comparison...”\n3. On page 6, Paragraph 3: “If Pi16+ fibroblasts are required for extravasation and immune infiltration, therapeutic exploitation...”\nSmall spelling mistakes 1. On page 5, Paragraph 1: “..., showing that that Pi16+ fibroblasts ...”, please delete the redundant “that”.\n2). On page 7 (Conclusion), Paragraph 2: “Indeed, whether Pi16 marks a lineage of fibroblasts... or experienced sheer-stress (Figure 1).” “sheer” should be “shear”.\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/13-126
https://f1000research.com/articles/13-761/v1	08 Jul 24	{ "type": "Systematic Review", "title": "The Role of Self-directed Learning in Promoting Deep Learning Processes: A Systematic Literature Review", "authors": [ "LI RUI", "NURFARADILLA MOHAMAD NASRI", "SITI NUR DIYANA” MAHMUD", "LI RUI", "SITI NUR DIYANA” MAHMUD" ], "abstract": "In current educational research, deep learning is widely considered a key approach to fostering the development of students’ comprehensive abilities. However, effectively promoting deep learning, especially across various educational settings, remains a challenge. Especially in the context of Self-Directed Learning (SDL) environments, current research does not specifically detail how SDL facilitates the deep learning process or how students experience and achieve deep learning within SDL environments. Addressing this research gap, this study explores the promotional effect of SDL on deep learning through a systematic literature review and analyzes how self-directed learning strategies and environments support the deep learning process of students. This article, based on Self-Determination Theory (SDT) and meta-cognitive theory, delves into the intrinsic factors and external conditions under SDL environments that promote deep learning. The research found that self-directed learning significantly promotes deep learning by fostering students’ active participation, self-management, and the development of metacognitive skills. Students’ interests and intrinsic motivation, along with reflective and evaluative activities, play a crucial role in deepening the understanding of knowledge. The effective use of technology provides the necessary support for self-directed learning, further facilitating students’ deep learning. Despite the close relationship between SDL and deep learning, effectively integrating these two modes of learning within today’s educational environment remains a challenge. The future requires educators to continue exploring innovative teaching methods and learning environments to promote the effective integration of SDL and deep learning, thereby improving the quality of education to meet future challenges.", "keywords": [ "self-directed learning", "deep learning", "metacognitive skills", "intrinsic motivation." ], "content": "Introduction\n\nDeep learning refers to learners’ profound understanding and application of knowledge. It plays a key role in helping students deeply understand and apply knowledge, adapt to future challenges, and promote personal comprehensive development, making it an indispensable part of contemporary education. Numerous studies have shown that deep learning requires students to undergo processes of reflection, critique, and the transfer and integration of knowledge. It focuses on cultivating students’ critical thinking and problem-solving abilities, emphasizes the capacity for students to “learn how to learn,” assists students in establishing habits and abilities for autonomous learning, and lays the foundation for lifelong learning (Hsieh & Maritz, 2023; Salleh et al., 2019; Sun et al., 2023; van der Graaf et al., 2022).\n\nSelf-Directed Learning (SDL) emphasizes the intrinsic proactivity of student learning (Lai et al., 2024) and highlights learners’ self-guidance and decision-making in the learning process (Evenhouse et al., 2023; Sun et al., 2023). SDL requires learners to actively seek resources and acquire knowledge based on their interests and needs. This proactivity facilitates learners’ in-depth exploration of learning topics and critical thinking about knowledge, effectively promoting deep learning among students (Lai et al., 2024; Salleh et al., 2019). By leading their learning and autonomously setting learning goals, evaluating and selecting learning resources, and self-monitoring the learning process, learners develop critical thinking and problem-solving skills (Adinda & Mohib, 2020; An & Qu, 2021; van der Graaf et al., 2022). These skills are also at the heart of students experiencing deep learning, as they foster the learner’s ability to recall, analyze, evaluate knowledge, and apply this knowledge in solving real-world problems (George et al., 2020; Nhat & Le, 2023).\n\nBoth SDL and deep learning promote learners’ profound understanding and application of knowledge (Aguiar-Castillo et al., 2021; Stephen & Rockinson-Szapkiw, 2021). By adopting self-directed learning approaches, learners can choose their learning content based on their interests and needs, making them more inclined to explore and delve into the subject matter. This facilitates achieving a deeper level of knowledge understanding and application, thus engaging in the process of deep learning (Aguiar-Castillo et al., 2021; Lai et al., 2024). However, despite ongoing updates in scholarly research on self-directed learning and deep learning, questions about how SDL methods can enhance the depth of students’ knowledge acquisition and application and how to foster the deep learning process in students remain for further investigation. Current research lacks studies on specific implementation strategies combining SDL with deep learning. Although theoretically, their integration holds great potential, how to effectively integrate the two in practice, especially across various educational settings, continues to be a challenge. While SDL and deep learning are widely discussed in the educational field, current studies often focus on them independently, lacking an understanding of their interplay and synergistic effects. Therefore, understanding how these two complement each other and promote individuals’ application of knowledge and skill enhancement is a challenging exploration area for educators and learners. Delving into this area can not only help learners better grasp complex concepts and skills but also pave the way for new educational models and learning methods, further promoting personalized learning and the realization of lifelong learning.\n\nGiven the current state of research, this study aims to analyze the interaction between self-directed learning and deep learning, exploring which factors can facilitate the occurrence of deep learning among university students within the process of self-directed learning and how students experience and achieve deep learning through self-directed learning. This aims to address the current gaps in research on the mechanisms of interaction between self-directed learning and deep learning, providing recommendations for higher education.\n\n\nLiterature review\n\nThe concept of Self-Directed Learning (SDL) originated from research into adult learning in the 1960s, and subsequently, the scope of SDL research has expanded to encompass all ages and educational levels. The initial focus was on recognizing adults’ conscious self-direction in learning and explaining how they learn (Merriam and Baumgartner, 2020). Houle (1961), through in-depth interviews with adult learners, categorized self-directed learning among adults into goal-oriented, learning-oriented, and activity-oriented types based on the motivation for participating in learning, providing a foundational framework for subsequent scholars’ research on adult learning. In 1966, Tough first introduced the concept of “self-directed learning,” whose connotation was further developed and expanded by Knowles (Tough, 1966). In 1975, Knowles published a seminal work on SDL, which is considered the most cited publication on SDL (Knowles, 1975).\n\nSince Knowles (1975), there has been a considerable effort to define and describe SDL. Currently, scholars tend to understand the essence of “Self-Directed Learning” from two perspectives: a process-oriented view and a personality trait view. Among them, the typical “process-oriented” perspective includes: Knowles posits that children are learners with a higher dependence, while adults are independent learners. This implies that adult learning does not rely predominantly on external guidance like that of children; instead, they can self-regulate, completing their learning based on self-direction. Knowles views individuals as autonomous and independent, which is both a prerequisite and foundation for adults’ self-directed learning. Long (2000) sees self-directed learning not just as an outcome, but as a process where the learner is responsible for initiating, planning, implementing, and monitoring their learning. Additionally, many scholars from a psychological standpoint regard self-directed learning as a personality trait or inclination, a “personality trait view”: Wiley (1983) defines “Self-Directed Learning Readiness (SDLR)” as “the degree to which an individual possesses the attitude, abilities, and personality characteristics necessary for self-directed learning.” Lounsbury (2009) and others argue that there is a logical relationship between self-directed learning and personality traits.\n\nThis study adopts the definition by Knowles (1975), positing that self-directed learning is a process in which learners, based on actual conditions, identify certain learning needs and, by learning objectives, formulate relevant learning plans. Subsequently, they autonomously implement these plans and ultimately evaluate the learning outcomes.\n\nCurrent research on Self-Directed Learning (SDL) involves multiple fields, such as lifelong learning, intrinsic motivation, online learning, blended learning, and deep learning. The growing domains in SDL research highlight its significant position in educational research and also indicate that educational researchers are increasingly recognizing the importance of cultivating learners’ self-directed learning capabilities. For example, research has demonstrated that SDL is a key characteristic of the capacity for lifelong learning and plays a crucial role in students’ academic and personal growth (Salleh et al., 2019). SDL not only promotes deep learning but also shows a significant correlation with academic achievement (Altinpulluk et al., 2023). This learning approach relies on students’ intrinsic motivation, self-efficacy, and willingness to face challenges. These factors work together to form the attitude and ability for self-directed learning in learners. Studies have shown that learners with high self-directed learning skills are more likely to become proactive self-regulators, exhibiting significant differences from lower achievers in terms of learning strategies, learning awareness, preparation, time, resource utilization, and peer support (van der Graaf et al., 2022). Research has confirmed a moderate positive correlation between self-directed learning and intrinsic motivation, indicating that learners’ intrinsic motivation is an important factor supporting self-directed learning (Altinpulluk et al., 2023). Educational practices, such as flipped classrooms, can effectively enhance students’ enthusiasm and self-directed learning capabilities (Hsieh & Maritz, 2023). Furthermore, learning styles impact students’ academic performance and the time invested in self-directed learning, with a variety of instructional guidance options provided by teachers further promoting certain aspects of students’ autonomous learning (Ganji et al., 2022; Hsieh & Maritz, 2023). Additionally, pedagogical and instructional design methods have potential impacts on fostering the development of students’ self-directed learning abilities in blended learning environments (Adinda & Mohib, 2020). Research indicates that SDL plays a key role in the deep processing of knowledge and is correlated with learning outcomes and academic achievements (Lai et al., 2024). Furthermore, creative learning outcomes can be supported through self-directed learning, and the attitudes and methods of self-directed learning positively predict online learning engagement. This positive relationship is mediated by the perceived value of learning goals, illustrating how self-directed learning fosters deeper levels of learning engagement (Sun et al., 2023). With the advancement of educational technology, research on SDL has also begun to focus on new trends in learning, such as Massive Open Online Courses (MOOCs) and home learning during the COVID-19 pandemic (Alhammadi, 2021; Altinpulluk et al., 2023). These studies not only explore the connection between SDL and global educational reforms but also emphasize the role of personal interest and self-regulation in different types of autonomous technology activities. Notably, personal interest is a significant predictor across all types of technological activities, while self-regulation primarily predicts instructional-oriented, information-oriented, and social-oriented activities (Lai et al., 2024). However, excessive cognitive load, especially in cases where cognitive abilities are not yet fully developed, may lead only to superficial changes in knowledge rather than deep learning (Butcher & Sumner, 2011). Therefore, educators need to consider this when designing and implementing self-directed learning activities to ensure that the activities can stimulate students’ interest without exceeding their cognitive load.\n\nIn summary, self-directed learning is a key factor in student success, promoting deep learning and academic achievement. However, which factors facilitate deep learning in the self-directed learning process, and how students experience and achieve the deep learning process require further study.\n\nDeep learning, as a significant topic in the field of education, has seen its understanding and definition evolve through various stages over time. From early studies to modern theoretical explorations, scholars from multiple perspectives have defined and interpreted deep learning, creating a rich series of theoretical frameworks and viewpoints. In 1976, Ference Marton and Roger Säljö first introduced the concepts of Surface Learning and Deep Learning. Through experimental research on students reading academic articles, they differentiated between two learning approaches: Surface Learning focuses on the superficial information and rote memorization of texts, whereas Deep Learning emphasizes understanding the deep meaning of texts, questioning the author’s viewpoints, and connecting with one’s knowledge and experience (Marton, F., & Säljö, R., 1976). Scholars gradually expanded on this concept. For example, John Biggs built on Marton’s work to propose that deep learning involves high-level or active cognitive processing (Biggs, 1979). In “7 Powerful Strategies for Deep Learning,” Eric Jensen and LeAnn Nickelsen further highlighted the multi-level processing in deep learning and the transformation in learners’ thoughts, behaviors, or control during the learning process (Jensen, E., & Nickelsen, L., 2008). In China, research on deep learning began around 2005. Li Jiahao and others defined deep learning as the process of understanding new ideas and facts critically and integrating them into the existing cognitive structure (He Ling, & Li Jiahao, 2005). Subsequently, scholars domestically and internationally conducted deeper research on deep learning, exploring and defining it from various angles, including learning methods, processes, and outcomes. Modern research on deep learning not only focuses on the depth of the learning process, emphasizing the transition from superficial memorization to deep understanding and creation, but also values the learning outcomes, namely the development of learners’ problem-solving capabilities, metacognitive abilities, and creative thinking skills (Weng et al., 2023). Scholars like Julian Hermida and the National Research Council (NRC) in the United States emphasize deep learning as a continuous process of knowledge construction, aiding learners in solving practical difficulties and issues in social interactions (Hermida, J., 2014; Council, N.R., 2013). Canadian scholar Michael Fullan and others have proposed a new pedagogical perspective, suggesting that deep learning involves new types of learning partnerships between students and teachers, digital tools and resources, and deep learning tasks (Fullan, M., & Langworthy, M., 2014).\n\nThis study posits that deep learning is a learning process driven by intrinsic motivation, critically integrating and deeply processing information from multiple sources. It aims to foster the development of higher-order cognitive abilities—including application, analysis, evaluation, and synthesis capabilities (derived from Bloom’s taxonomy)—to achieve problem-solving and knowledge transfer. Furthermore, deep learning also emphasizes cultivating learners’ core competencies, such as collaboration, communication, autonomous learning, perseverance in learning, critical thinking, and innovative thinking, to meet the complex demands of future society. Through this process, learners can profoundly understand the content and effectively apply knowledge in new contexts, thereby promoting comprehensive personal development on multiple dimensions.\n\nCurrent research on deep learning in the educational field primarily focuses on the study of learning strategies, the application of technologies and tools, and aspects such as student engagement and motivation (Aguiar-Castillo et al., 2021; Alhammadi, 2021; Fouché, 2024; Pereira & Wahi, 2021). For instance, some studies are dedicated to exploring and validating which learning strategies can effectively promote deep learning, such as critical thinking, problem-solving abilities, and reflective learning (Nhat & Le, 2023; Weng et al., 2023). Additionally, with the application of technology in education, researching how to use digital tools and online platforms to promote deep learning has become a hotspot. This includes exploring how blended learning, flipped classroom models, and gamified learning can support the deep learning process (An & Qu, 2021; Pereira & Wahi, 2021).\n\nThere are also studies dedicated to how to increase student engagement and intrinsic motivation during the deep learning process. This involves how to design learning activities that stimulate students’ curiosity and desire to explore, as well as how to enhance students’ self-efficacy and satisfaction with learning through autonomous learning strategies (Aguiar-Castillo et al., 2021; Pereira & Wahi, 2021). As the educational community deepens its understanding of student autonomy and deep learning, an increasing number of studies emphasize the critical role of autonomous learning in promoting deep learning. Research is also increasingly highlighting the integration of theory and practice, as well as the combination of knowledge from different disciplines (Parpala et al., 2022; Segú Odriozola, 2023). Based on current research, the integration of deep learning and self-directed learning (SDL) remains relatively unexplored. Although deep learning is considered a key approach to fostering students’ comprehensive skill development, its potential when combined with SDL has not been fully investigated. Self-directed learning emphasizes learner initiative and autonomy, aligning with the active participation emphasized in deep learning. Deep learning also emphasizes promoting students’ active exploration of knowledge and fostering students’ autonomy in learning. Therefore, focusing research on how to effectively integrate and apply these two modes of learning to optimize educational strategies and improve learning outcomes is both feasible and necessary. Future research on deep learning needs to pay more attention to how the principles of deep learning can be combined with self-directed learning strategies. In this way, educators can better meet students’ individualized and deep learning needs, promoting comprehensive student development.\n\n\nTheoretical framework\n\nSelf-Determination Theory (SDT) was originally proposed by Deci and Ryan. SDT emphasizes three fundamental psychological needs: Autonomy, Competence, and Relatedness (Deci & Ryan, 1985). SDT posits that if these three basic psychological needs are not met, an individual cannot grow and progress. The three core psychological needs of Self-Determination Theory provide an important perspective for understanding and researching Self-Directed Learning (SDL) and deep learning. Guided by SDT theory, research can better understand why students’ self-directed learning behavior can effectively promote deep learning. When students exhibit strong autonomy in their learning, it is often accompanied by strong intrinsic motivation, leading them to more actively engage in the learning process, explore knowledge, and deepen their understanding and application of knowledge. At the same time, the satisfaction of competence increases students’ sense of achievement during the learning process, enhancing their willingness to continue exploring and solving complex problems. The fulfillment of relatedness needs strengthens the connection and interaction between students and their teachers and peers, creating a supportive social environment for deep learning.\n\nMetacognitive theory focuses on individuals’ cognition, monitoring, and regulation of their learning processes (Flavell, J. H., 1976). It comprises three main components: metacognitive knowledge, metacognitive monitoring, and metacognitive experiences (Li Hongyu & Yin Hongxin, 2004). Metacognitive abilities enable learners to effectively plan, monitor, and evaluate their cognitive processes, thus better controlling the learning process. This study analyzes how autonomous learning activities promote the development of students’ metacognitive abilities, thereby supporting deep learning, under the guidance of metacognitive theory. By exploring how autonomous learning strategies (such as goal setting, self-monitoring, reflection, etc.) help students enhance their cognition and regulation of their learning processes, the research reveals the specific impact of these strategies on facilitating deep learning.\n\nSelf-Determination Theory (SDT) and Metacognitive Theory jointly provide strong theoretical support for this study. SDT analyzes the impact of psychological need satisfaction on the motivation for deep learning, while Metacognitive Theory explores how self-monitoring and regulation of the learning process promote deep learning abilities. Together, they offer theoretical and methodological guidance for examining the facilitative role of self-directed learning approaches in the deep learning process.\n\n\nResearch questions\n\n\n\n1. What is the interrelationship between Self-Directed Learning and Deep Learning?\n\n2. During the process of Self-Directed Learning among university students, which factors are most conducive to the occurrence of Deep Learning?\n\n3. How do students experience and achieve Deep Learning within Self-Directed Learning?\n\n\nMethods\n\nThrough conducting a systematic literature review and meta-analysis, this study aims to collect, evaluate, and integrate evidence on the interaction between Self-Directed Learning and Deep Learning from existing literature, to identify and analyze key Self-Directed Learning conditions and factors that facilitate Deep Learning.\n\nTo effectively obtain the target literature for this study, a precise title search was conducted in the Web of Science database using keywords such as “Self-Directed Learning,” “Self-Regulated Learning,” “Independent Learning,” “Deep/Deeper Learning,” “Deep Approach,” “Deep Processing,” “Deep Strategies,” and “Deep Learner.” The literature search was limited to publications between January 1, 2020, and March 8, 2024, with the final search conducted on March 19, 2024, yielding a total of 265,092 articles (Deep Learning n=253,980, Self-Directed Learning n=2,112). The Web of Science database was chosen for this literature review because it is a renowned academic resource database that includes high-quality, peer-reviewed journal articles, conference papers, and more from various fields. It is widely recognized as an essential academic resource for the sciences, social sciences, arts, and humanities.\n\nTo ensure the accuracy and reliability of the literature analysis results and to precisely present the interrelationship between Self-Directed Learning and Deep Learning, as well as the research situation regarding the facilitative role of Self-Directed Learning on Deep Learning, based on the research questions, this study established literature inclusion/exclusion criteria for the initially retrieved 265,092 documents as shown in Table 1. The first six criteria in Table 1 are common standards for selecting literature in systematic reviews, the seventh criterion restricts the research scope to higher education, the eighth criterion specifies the quality of the literature, excluding documents lacking a research question, rigorous research process, and clear research methods; the ninth criterion aims to focus the literature’s research topic on the study of Self-Directed Learning and Deep Learning, excluding research literature whose focus does not align.\n\nThis study follows the methodology of a systematic literature review and meta-analysis, adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for literature selection. We can visit the link (https://doi.org/10.6084/m9.figshare.26054176) for a complete list of reports. The screening process for the initial retrieval of 265,092 articles is as follows:\n\n1. Excluded 128,218 articles with inaccessible full texts (SDL n=942; Deep Learning n=127,276).\n\n2. Excluded 336 non-English papers (SDL n=39; Deep Learning n=297).\n\n3. Excluded 24,245 non-journal papers (SDL n=118; Deep Learning n=24,127).\n\n4. Excluded 102,315 papers unrelated to the topic (SDL n=544; Deep Learning n=101,771).\n\nThis left 1,978 articles (SDL n=469; Deep Learning n=1,509) for abstract and full-text review. Subsequently, a secondary search was conducted using a snowballing technique, resulting in 87 additional relevant papers (SDL n=38; Deep Learning n=49). After full-text review, 952 papers were excluded for being unrelated, non-educational, or non-empirical (SDL n=462; Deep Learning n=490).\n\nThe remaining 113 papers (SDL n=45; Deep Learning n=68) were then scrutinized for research questions, methodology, and conclusions, leading to the exclusion of 74 papers with unclear research questions, methodologies, or conclusions (SDL n=31; Deep Learning n=43). Ultimately, 39 papers were included in the final analysis (SDL n=14; Deep Learning n=25). The specific screening process is illustrated in Figures 1 and 2 (The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram can also be viewed via the link: https://doi.org/10.6084/m9.figshare.26114413 (Rui, 2024c), with the number of included papers detailed in Table 2.\n\nOur research team consists of three members. The article collection process was conducted by one member, and the collected articles were independently evaluated for quality by two experts in the team using the Joanna Briggs Institute (JBI) checklist. Based on the review results, all reviewers unanimously agreed that 39 articles met the minimum quality standards required for this study, and any discrepancies in evaluation judgments were resolved.\n\nThe data items included the article title, authors, publication year, host country, abstract, research topics, research methods (qualitative, quantitative, mixed methods, etc.), participant characteristics (age, educational background, etc.), study outcomes, sample size, research tools, research findings, contributions to deep learning research, contributions to self-directed learning research, student learning experiences, learning strategies, and deep learning outcomes. Through a thorough reading of the articles, the researchers manually organized the aforementioned information and summarized the data from the 39 articles into a series of tables. After a series of meetings and discussions, the three authors revised and refined the table data to minimize potential data bias in the research process.\n\nThe data analysis process of this study went through three stages: key information summary, dimensional analysis, and content analysis.\n\nThe first phase involved summarizing the key information of the literature. The method of the content summary was utilized to distill the main content, research themes, findings, and contributions of the literature, allowing for a quick understanding of the core content and research focus, thereby laying the foundation for subsequent in-depth analysis. During this stage, the following tables were formed: Tables 3, 4, 5, 6, 7, 8. The exact details of the table can be found in the extended data (Rui, 2024b).\n\nThe second phase involved a dimensional analysis of the literature. Using thematic analysis, key concepts and themes were extracted and identified from the literature to explore what kind of relationship exists between SDL and deep learning, how SDL facilitates deep learning, and what are the key factors and conditions in SDL that promote deep learning. A deeper understanding of the interaction and influence mechanisms between SDL and deep learning was achieved through the categorization and comparison of key concepts and themes appearing in the literature. During this stage, the following tables were formed: Tables 9, 10. The exact details of the table can be found in the extended data (Rui, 2024b).\n\nThe third phase is content analysis. During the content analysis phase, the focus is on exploring how students experience and achieve deep learning within Self-Directed Learning. The analysis at this stage aims to deeply understand students’ learning experiences, the learning strategies they adopt, and how these strategies facilitate the achievement of deep learning. Content analysis, through a detailed examination of case studies, empirical research findings, and theoretical discussions in the literature, distills key experiences and processes that contribute to deep learning. During this stage, the following tables were formed: Tables 11, 12. The exact details of the table can be found in the Figshare system. We can access the link (https://doi.org/10.6084/m9.figshare.26012824) to get the analyze process data.\n\n\nResults and Discussion\n\nKey Processes in Self-Directed Learning Strategies and Environments that Facilitate Deep Learning\n\nSelf-directed learning emphasizes the learner’s active participation and self-management abilities, which directly support key processes in deep learning, such as active exploration, critical thinking, and the ability to integrate new knowledge with existing knowledge (Long, H. B., 2000). For example, in online learning environments, students facilitate deep understanding and application of knowledge by autonomously selecting learning resources, setting their learning objectives, and managing their time (Sun et al., 2023). Research indicates that an individual’s interest in learning topics and the tendency to pursue personal interests through learning are key drivers for engaging students in self-directed learning (Lai et al., 2024). Interest and intrinsic motivation, as integral components of self-directed learning, are equally crucial to the occurrence of deep learning. Interest and intrinsic motivation can stimulate students’ curiosity and desire to explore, thereby facilitating the learning of complex concepts (Litzinger et al., 2005). Additionally, reflective learning activities within a self-directed learning environment, such as learning logs and self-assessment, can encourage students to deeply reflect on their learning processes and outcomes (Stephen & Rockinson-Szapkiw, 2021). This reflection not only helps students identify and correct misconceptions in their learning but also promotes a deep understanding of knowledge and the formation of long-term memory. Research indicates that environments supporting self-regulated learning behaviors are particularly important for deep learning (Evenhouse et al., 2023). For example, the blended model of autonomous learning and teacher-guided learning provided by blended learning environments, as well as the features of online course platforms that support students’ autonomous learning, are effective conditions for facilitating deep learning (Onah et al., 2020). During the self-directed learning process, the effective use of technology, such as online resources and Learning Management Systems (LMS), enhances the flexibility and convenience of accessing information, supports students in deeply exploring knowledge based on personal interests and needs, and further facilitates the occurrence of deep learning (Fanshawe & Barton, 2023).\n\nDeep Learning, in Turn, Promotes the Practice and Development of SDL\n\nThe deep understanding and application of knowledge within the deep learning process can ignite learners’ curiosity and thirst for knowledge, thereby enhancing their interest in exploring new knowledge. This sustained learning motivation is a key factor for the success of SDL (Hsieh & Maritz, 2023). Deep learning aids learners in building a more solid and systematic knowledge structure by fostering critical thinking, problem-solving abilities, and the application of knowledge (Weng et al., 2023). This efficient learning process can enhance an individual’s self-directed learning capabilities, enabling learners to more effectively plan, monitor, and adjust their learning pathways (Ma, 2022). Reflective and evaluative activities in the deep learning process promote a profound understanding of one’s learning process, including recognizing one’s learning style, strengths, and areas for improvement (Nhat & Le, 2023). This self-reflection and evaluation capability is a core component of SDL, essential for learners to autonomously adjust their learning strategies (Fanshawe & Barton, 2023). Through engaging in deep learning activities, learners can more flexibly adapt to complex and uncertain learning situations, selecting appropriate learning resources and strategies. This adaptability and flexibility are necessary conditions for implementing effective SDL (Evenhouse et al., 2023; Onah et al., 2020; Stephen & Rockinson-Szapkiw, 2021).\n\nIn summary, deep learning is not only a result of SDL but also provides strong support for the implementation of SDL. Through deep learning, learners can cultivate a more proactive, reflective, and collaborative learning attitude, thereby further strengthening and optimizing the process of self-directed learning.\n\nThe potential connection between Self-Directed Learning (SDL) and Deep Learning\n\nThe reciprocal relationship between Self-Directed Learning (SDL) and Deep Learning indeed forms the core of their interaction, but there are also other potential connections between the two. These connections are not limited to direct mutual promotion but also include the wider educational context and the impact on personal development levels.\n\nCommon promoters of cognitive development: Both SDL and deep learning are committed to advancing learners’ cognitive development, especially in promoting higher-order thinking skills such as critical thinking, analysis, and evaluation. The development of these skills aids learners in thinking and making decisions more independently and effectively when faced with diverse and complex problems (Altinpulluk et al., 2023; Hsieh & Maritz, 2023; Nhat & Le, 2023; Saqr, Matcha, Jovanovic, et al., 2023).\n\nThe foundation of lifelong learning: Existing research has demonstrated that both SDL and deep learning are key components of lifelong learning. In today’s era, where knowledge is updated at an increasingly rapid pace, individuals must continually learn new knowledge and skills to adapt to changes in the times. SDL emphasizes the autonomy and initiative of learning, while deep learning values the quality and durability of learning. Combined, they provide a solid foundation for lifelong learning (Alhammadi, 2021; Geitz et al., 2023; Salleh et al., 2019; Saqr, Matcha, Jovanovic, et al., 2023).\n\nThe interplay of affect and motivation: The sense of achievement and sustained interest in exploring learning content generated during the deep learning process can significantly enhance learners’ intrinsic motivation. Intrinsic motivation not only drives learners to engage in deeper learning processes, but it is also crucial for the sustainability and effectiveness of SDL. Additionally, the enhancement of self-efficacy in SDL, in turn, strengthens learners’ motivation to engage in deep learning (Altinpulluk et al., 2023; Hsieh & Maritz, 2023; Lai et al., 2024; Nhat & Le, 2023), which not only improves the effectiveness of learning but also increases its sustainability.\n\nTo address this issue, the study first conducted a thematic analysis of the literature, identifying key concepts and themes from the documents, and summarizing the essential information in Table 9. Specific information for Table 9 can be found in the attachment. Based on the information in the table, the study further merged related themes, reducing redundant information while improving conceptual clarity to more effectively identify key factors. At this stage, Table 10 was generated: Analysis of Analysis table of key factors for the self-directed learning environment to promote deep learning. Table 10 displays the key factors that promote deep learning in the process of self-directed learning, including two main categories: internal factors and external conditions. Internal factors include self-regulation and self-directed learning abilities, interest and motivation, metacognitive skills, as well as reflection and evaluation. External conditions involve the effective use of technology, interactive learning environments, innovative teaching models, and situational engagement and gamification. Each key factor will be analyzed next.\n\nInternal factors\n\nSelf-regulation and self-directed learning abilities: Self-regulation and self-directed learning abilities are widely regarded as key elements for deep learning, capable of effectively facilitating the occurrence of deep learning among students. For example, the paper by Stephen & Rockinson-Szapkiw (2021) enhances students’ self-regulation and self-direction abilities through the design and implementation of a first-semester seminar course, particularly emphasizing the role of reflective activities (such as learning logs). Through continuous participation and reflection on the learning process, it promotes a deep understanding and application of knowledge. Similarly, the paper by Onah et al. (2020), by embedding MOOC platforms, promotes autonomous learning and self-regulated learning skills among undergraduate students, demonstrating how self-directed learning can facilitate deep learning within the context of integrating traditional and modern educational backgrounds.\n\nInterest and motivation: Individual interest and intrinsic motivation, as significant factors driving deep learning, have a positive effect on students’ deep learning. For example, (Lai et al., 2024) explored how individual interest and self-regulation interact and affect the process of autonomously using technology for language learning, highlighting the critical role of individual interest and intrinsic motivation in promoting deep learning.\n\nMetacognitive skills: Students’ metacognitive awareness is considered a key factor for deep learning. Metacognitive abilities, including metacognitive monitoring and regulation, enable students to monitor and reflect on their learning processes, identify learning obstacles, and effectively adjust their learning strategies (Moonen-Van Loon et al., 2022). Onah et al. (2020) facilitated students’ autonomous learning and self-regulated learning skills through the use of blended learning environments and MOOC platforms, reflecting the role of metacognitive skills in planning, executing, and evaluating learning strategies. Hua & Wang, (2024) research on the use of Learning Management Systems (LMS) supported students in self-monitoring during the learning process and allowed students to assess their learning progress, emphasizing the importance of metacognitive skills in enhancing deep learning and autonomous learning abilities.\n\nReflection and evaluation: Reflective learning activities such as learning logs and self-assessment play a very important role in deep learning. These methods help students reflect on their learning processes and the learning strategies applied, constituting a key component of deep learning (Stephen & Rockinson-Szapkiw, 2021). Students developed self-regulation and self-directed abilities in the process of completing reflective activities in learning logs, promoting a deep understanding and application of knowledge (Fanshawe & Barton, 2023). Research utilizing text mining technology to assist with feedback interpretation helped students self-assess and formulate new learning goals and strategies, emphasizing the role of reflection and evaluation in deep learning.\n\nExternal factors\n\nEffective use of technology: Research has demonstrated that technology plays a positive partial mediating role between autonomous learning and lifelong learning (Salleh et al., 2019). Interaction, sharing, and collaborative activities conducted through social networks can facilitate communication among students, knowledge sharing, and deep understanding (Salleh et al., 2019). In the current educational context, technological proficiency and the effective use of online resources are considered key to promoting deep learning. (Lai et al., 2024) The interaction between individual interest and self-regulation in the process of using technology for language learning demonstrates how technology supports personalized and deep learning. Autonomous informal learning using online resources highlights the role of technology in providing a learning environment with choices and freedom. (Salleh et al., 2019) the study proves that the effective use of technology can promote students’ deep learning.\n\nInteractive learning environments: Research has shown that diverse and interactive learning environments (such as flipped teaching) can effectively increase student engagement and motivation for learning. Students’ perceptions of the learning environment, including the accessibility of resources, the supportiveness of the platform, and interaction with teachers and peers, are crucial to deep learning (Geitz et al., 2023; Thompson & Lake, 2023; Evenhouse et al., 2023). The blended learning environment provides students with diverse learning resources and supports self-regulated learning behaviors, offering the necessary conditions and opportunities for deep learning to occur, and enabling learners to engage in in-depth study. The introduction of interactive learning environments strengthens this process by promoting deeper levels of thinking, understanding, and application, thus more effectively facilitating the occurrence of deep learning (Salleh et al., 2019).\n\nInnovative teaching models: Innovative teaching encompasses the adoption of innovative instructional designs and curricular structures, such as Project-Based Learning (PBL) and problem-solving, to provide authentic and challenging learning experiences. These methods encourage students to actively explore and apply knowledge, fostering the development of critical thinking and innovative capabilities. The study by Tuononen et al., (2023) highlights the effectiveness of innovative teaching in promoting deep learning through case-based instruction that provides authentic learning contexts, encourages active exploration and application of knowledge, develops higher-order thinking skills, and facilitates the internalization and transfer of knowledge. The study by Geitz et al., (2023) discusses how Design-Based Education (DBE) in foundational education, by providing a learning environment aligned with vocational fields and emphasizing the integration of practice and theory, enables students to engage in deep learning while solving real-world problems and developing key 21st-century skills.\n\nSituational engagement and gamification: Contextual engagement and gamification effectively enhance the enjoyment and interactivity of learning, significantly increasing students’ emotional and behavioral engagement. Through the enhancement of student participation, they promote deeper learning and improved learning outcomes. These strategies, by creating engaging learning environments and practical opportunities, enhance students’ motivation to learn and sense of participation, thereby facilitating deep learning and the effective application of knowledge. Additionally, research by Salleh et al., (2019) demonstrates that informal autonomous learning utilizing online resources emphasizes that an individual’s interest in the learning topic is the primary driver of deep learning and emotional engagement. Research by Altinpulluk et al., (2023) has demonstrated that there is a positive relationship between self-directed learning and levels of intrinsic motivation within MOOC environments. The study also highlights the role of contextual engagement and gamification strategies in enhancing motivation and deep learning.\n\nIn the process of self-directed learning, these internal factors and external conditions work together through various mechanisms to facilitate the occurrence of deep learning, such as enhancing learning motivation, providing abundant learning resources, promoting communication and cooperation among students, and developing critical thinking and problem-solving skills. Taking these factors into account, it is possible to create a rich and diverse learning environment that is conducive to deep learning for students.\n\nBefore answering how students experience and achieve deep learning within the process of self-directed learning, a content analysis of the literature is first conducted to summarize and categorize specific experiences of students during self-directed learning and how these experiences facilitate the achievement of deep learning. This includes the deep learning strategies adopted, and how specific learning experiences promote a deep understanding and application of knowledge. Detailed content is shown in Table 11: Table of Students’ Self-Directed Learning Experiences and Strategies for Achieving Deep Learning. Further content analysis of the literature is then conducted to delve deeper into the outcomes of deep learning achieved through the strategies or activities implemented during the process of self-directed learning. This analysis explores how learning strategies or activities specifically impact students’ outcomes in deep learning, including changes at the cognitive, emotional, and behavioral levels. Detailed content is shown in Table 12: Table of Achievements of Deep Learning in the Process of Students’ Self-Directed Learning.\n\nThrough thematic analysis of the content of the tables, it was discovered that students achieve deep learning during the process of self-directed learning through a series of strategies and activities. These can be summarized as reflection and self-regulation, enhancement of intrinsic and extrinsic motivation, utilization of technology and social media, Problem-Based Learning (PBL), and interdisciplinary collaboration, as well as the cultivation of metacognitive awareness.\n\nReflection and self-regulation\n\nReflection and self-regulation are key mechanisms for achieving deep learning in the process of self-directed learning. By writing learning logs and engaging in reflective writing activities, students can not only examine and evaluate their learning approaches, understanding processes, and their effectiveness but also identify and overcome obstacles in learning. Reflection and self-regulation are key mechanisms for achieving deep learning in the process of self-directed learning. By writing learning logs and engaging in reflective writing activities, students can not only examine and evaluate their learning approaches, understanding processes, and effectiveness but also identify and overcome obstacles in learning. Continuous self-monitoring and adjustment in learning not only directly affect the depth and efficiency of learning but also facilitate the development of students into independent and self-driven learners. Research by Stephen & Rockinson-Szapkiw (2021) indicates that engaging in high-impact educational practices, such as learning logs and reflective writing, can significantly enhance students’ self-regulation skills and promote a deeper understanding and application of knowledge. The study by Fanshawe & Barton (2023)found that supporting doctoral students’ self-directed learning through the use of a Learning Management System (LMS) revealed that regular reflection and self-monitoring activities help deepen students’ understanding of academic research and can assist in enhancing their academic writing and research skills. Current research underscores the importance of self-regulated learning abilities for deep learning. Through the integrated use of reflective learning activities such as learning logs and reflective writing, along with the cultivation of self-regulated learning abilities, students can achieve a deeper understanding and application of knowledge in the process of self-directed learning. The development of these strategies and skills is beneficial not only for current learning tasks but also lays a solid foundation for students’ lifelong learning.\n\nEnhancement of intrinsic and extrinsic motivation\n\nIn the process of self-directed learning, the enhancement of both intrinsic and extrinsic learning motivation is crucial for promoting deep learning. The enhancement of learning motivation encourages students to participate more actively in learning and to explore knowledge more proactively, thus achieving a deeper understanding and application of knowledge. The study by Hsieh & Maritz (2023) detailed how flipped classroom instruction enhances students’ active participation in class, thereby improving their intrinsic motivation and self-directed learning abilities. Additionally, this model supports students in adjusting their learning pace according to their speed, aiding in the development of their self-regulated learning capabilities and lifelong learning habits. The study by Aguiar-Castillo et al. (2021) shows that by integrating gamification elements into the learning process in higher education, students are encouraged to explore the unknown. By solving challenges and puzzles within games, students’ curiosity and desire to explore are sparked, promoting active learning. In the process of exploration, students not only acquire new knowledge but also learn how to learn, cultivating critical thinking abilities and the development of higher-order skills.\n\nUtilization of technology and social media\n\nThe utilization of social media platforms and online learning resources provides students with opportunities for interaction and collaboration. These platforms enable students to access a wide variety of learning materials. Digital learning methods not only greatly expand students’ knowledge and perspectives but also, through participation in online discussion groups and collaborative learning activities, allow students to share viewpoints, exchange ideas, and critically examine issues from different angles. Gradually, students develop the ability to deeply understand complex concepts and skills. Furthermore, this learning model also stimulates students’ critical thinking skills, enabling them to effectively discern and select when confronted with information. The study by Salleh et al. (2019) explores how social networking sites act as tools for self-directed learning, facilitating both individual and collective learning. It enhances learners’ control over their learning processes while also increasing the interactivity and cooperativeness of learning. The paper by Alhammadi (2021) analyzes how, during the COVID-19 pandemic, the quality of learning was maintained and enhanced through the use of online learning tools and platforms, especially social media. In a remote learning environment, the effective utilization of technology and social media plays a crucial role in maintaining student engagement, facilitating communication and collaboration, and supporting deep learning.\n\nProblem-Based Learning (PBL) and Interdisciplinary collaboration\n\nStudents, by applying and integrating interdisciplinary knowledge in multidisciplinary team collaborations to solve real-world problems, not only enhance their problem-solving and analytical abilities but also deepen their understanding of knowledge. Through the practical application of knowledge to solve specific problems, students can learn and understand disciplinary knowledge more deeply, thereby achieving deep learning. The study by Geitz et al. (2023) emphasizes the importance of Problem-Based Learning (PBL) and multidisciplinary collaboration in Design-Based Education (DBE) environments. DBE environments, by integrating real-world problems and multidisciplinary knowledge, foster deep learning among students, showing significant advantages, particularly in promoting students’ innovative and critical thinking. The study by Saqr, Matcha, Jovanovic, et al. (2023) indicates that effective learning strategies, especially when applied in PBL environments, are crucial for promoting deep learning. By transferring and applying these strategies across different learning contexts, students are better able to adapt to PBL environments, facilitating the integration and application of interdisciplinary knowledge.\n\nCultivation of metacognitive awareness\n\nThrough metacognitive activities, students learn how to effectively plan their learning processes, including setting clear learning objectives and selecting appropriate learning resources, monitoring their learning progress, and evaluating the effectiveness of their learning outcomes and strategies, thereby managing and guiding their learning more effectively. The cultivation of metacognitive awareness is a core component of deep learning because it involves students’ cognition and regulation of their learning process, including setting goals, choosing strategies, monitoring progress, and reflecting on learning outcomes. Through metacognitive activities, students are not only able to identify and utilize effective learning strategies but can also adjust their learning methods when faced with challenges, ensuring the achievement of learning objectives. The study by Tuononen et al. (2023) explores the role of metacognitive awareness in promoting deep learning. The research found that by cultivating students’ metacognitive awareness, their learning outcomes can be significantly improved, as students learn how to effectively plan, monitor, and regulate their learning processes. The study by van der Graaf et al. (2022) emphasizes the importance of metacognitive activities in self-regulated learning. By implementing cognitive and metacognitive activities to monitor the learning process, students can better achieve their learning objectives, demonstrating how metacognitive activities support the key processes of deep learning. Therefore, the implementation of cognitive and metacognitive activities not only helps students effectively monitor and regulate their learning process but is also a key strategy in driving them toward achieving deep learning objectives, significantly enhancing the quality and effectiveness of learning.\n\nTeaching strategies and methods\n\nReinforce reflection and self-regulation mechanisms: Incorporating regular reflective and self-assessment activities into course design, such as learning logs, project reflection reports, and self-assessment questionnaires, can help students engage in more effective self-regulation and reflection during self-directed learning, thereby promoting a deep understanding and application of knowledge.\n\nEnhance students’ intrinsic and extrinsic motivation: Teachers can adopt various teaching strategies based on the characteristics of the teaching content to motivate students’ extrinsic motivation while ensuring that learning activities are connected to students’ personal interests and career goals to enhance intrinsic motivation. The enhancement of motivation helps increase students’ enthusiasm and curiosity for learning, which is crucial for deep learning.\n\nPromote Problem-Based Learning (PBL) and interdisciplinary collaboration: Teachers can design PBL projects centered around real-world challenges, encouraging students to engage in interdisciplinary collaboration and autonomously seek solutions, providing resources and support when necessary, to cultivate students’ self-directed learning awareness and deep learning abilities.\n\nCultivate metacognitive awareness: Teachers can instruct students on how to effectively plan, execute, monitor, and adjust their learning through relevant courses and activities. Students are encouraged to apply these skills to enhance the effectiveness of self-directed learning and deep learning.\n\nAdopt innovative teaching and assessment methods: Develop and implement innovative teaching methods and assessment strategies, such as project-based learning, contextual learning, and flipped classrooms, to promote students’ deep learning. The design of assessment methods should consider students’ self-directed learning experiences and deep learning outcomes, ensuring that assessment methods accurately reflect students’ learning progress and achievements.\n\nLearning Environment and Support\n\nEffectively utilize technology and social media: Actively integrate technological tools and social media platforms to support the autonomy, collaboration, and interactivity of learning. Provide training and guidance to help students effectively use technological tools for information retrieval, knowledge sharing, and collaborative learning.\n\nCreate supportive and interactive learning environments: Build an open, supportive, and interactive learning environment that encourages effective communication and collaboration among students and between students and teachers. Utilize online discussion boards, peer reviews, and group projects to foster this interaction.\n\nBy implementing the above recommendations, educators can more effectively support students in achieving deep learning during the process of self-directed learning, promote students’ personal and professional development, and lay a solid foundation for students’ lifelong learning journey.\n\n\nConclusion\n\nThis review, through a systematic analysis of 39 studies, aims to reveal the role of SDL (Self-Directed Learning) in deepening students’ understanding and application of knowledge. The conclusion of this study summarizes the interaction between SDL and deep learning, the key conditions that facilitate the occurrence of deep learning in self-directed learning environments, and how students achieve deep learning during the SDL process.\n\nFirstly, Self-Directed Learning has been identified as a key strategy in promoting deep learning, especially in terms of enhancing student engagement, motivation, and personalized learning. SDL encourages students to choose their learning paths based on their interests, needs, and goals. This proactivity and autonomy are the cornerstones of deep learning. Through self-regulated learning strategies, the development of metacognitive awareness, and reflection on the learning process, students can more effectively plan, monitor, and adjust their learning activities, promoting a deep understanding and application of knowledge.\n\nSecondly, this study identifies multiple key conditions that facilitate the occurrence of deep learning among university students in the process of self-directed learning, from two dimensions: internal factors and external conditions. These include the utilization of technology and online resources, interest and motivation, metacognitive skills, self-regulation, and the application of evaluation and feedback mechanisms. These conditions provide the necessary support and resources for students’ deep learning, helping students achieve deep learning in a self-directed learning environment.\n\nFinally, students experience and achieve deep learning in the SDL process by adopting autonomous learning strategies, reflecting on and evaluating their learning processes, enhancing learning motivation, and making effective use of technology and media. These strategies and activities promote the development of students’ critical thinking, problem-solving abilities, and innovative thinking, laying a solid foundation for their future academic and professional careers.\n\nIn summary, self-directed learning plays a crucial role in promoting deep learning. By providing students with a supportive learning environment, abundant resources, and positive interaction opportunities, educators can foster students’ self-directed learning abilities, thereby deepening their understanding and application of knowledge. With the continuous development of educational technology, the integration of self-directed learning and deep learning will provide students with more personalized and flexible learning opportunities, further promoting their holistic development.\n\nEthical approval and consent were not required.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: The role of self-directed learning in promoting deep learning processes: a systematic literature review, Figshare. https://doi.org/10.6084/m9.figshare.26012824 (Rui, 2024b).\n\nThis project contains the following extended data:\n\n- Table Data Analysis Process. doc.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nRepository name: PRISMA checklist used for the systematic literature review, https://doi.org/10.6084/m9.figshare.26054176 (Rui, 2024a).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAdinda D, Mohib N: Teaching and instructional design approach to enhance students’ self-directed learning in blended learning environments. Electronic Journal of eLearning. 2020; 18(2): 162–174.\n\nAguiar-Castillo L, Clavijo-Rodriguez A, Hernández-López L, et al.: Gamification and deep learning approaches in higher education. J. Hosp. Leis. Sport Tour. Educ. 2021; 29: 100290. Publisher Full Text\n\nAlhammadi S: The effect of the COVID-19 pandemic on learning quality and practices in higher education—using deep and surface approaches. Educ. Sci. 2021; 11(9): 462. Publisher Full Text\n\nAltinpulluk H, Kilinc H, Alptekin G, et al.: Self-directed learning and intrinsic motivation levels in MOOCs. Open Praxis. 2023; 15(2): 149–161. Publisher Full Text\n\nAn X, Qu C: A hierarchical learning model based on deep learning and its application in a SPOC and flipped classroom. Int. J. Emerg. Technol. Learn. 2021; 16(9): 76–93. Publisher Full Text\n\nBiggs JB: Individual differences in study processes and the quality of learning outcomes. High. Educ. 1979; 8: 381–394.\n\nButcher KR, Sumner T: Self-directed Learning and the Sensemaking Paradox. Hum. Comput. Interact. 2011; 26(1-2): 123–159.\n\nClack A, Dommett EJ: Student learning approaches: Beyond assessment type to feedback and student choice. Educ. Sci. 2021; 11(9): 468. Publisher Full Text\n\nCouncil, N. R: Education for life and work: Developing transferable knowledge and skills in the 21st century. Washington, DC: National Academies Press; 2013.\n\nDeci EL, Ryan RM: Intrinsic motivation and self-determination in human behavior. New York: Plenum Press; 1985.\n\nEllis RA, Bliuc AM, Han F: Challenges in assessing the nature of effective collaboration in blended university courses. Australas. J. Educ. Technol. 2021; 37(1): 1–14.\n\nEvenhouse D, Lee Y, Berger E, et al.: Engineering student experience and self-direction in implementations of blended learning: a cross-institutional analysis. Int. J. STEM Educ. 2023; 10(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFanshawe M, Barton G: PhD by LMS: Using a learning management system to facilitate self-directed learning in a doctoral study. Australas. J. Educ. Technol. 2023; 39(4): 104–126. Publisher Full Text\n\nFlavell JH: Metacognitive aspects of problem solving. The nature of intelligence. Routledge; 1976; pp. 231–236.\n\nFouché I: Pre-service teacher investment through dialogic action learning. Reading & Writing-Journal of the Reading Association of South Africa. 2024; 15(1): 452. Publisher Full Text\n\nFullan M, Langworthy M: A rich seam: How new pedagogies find deep learning. London: Pearson; 2014.\n\nGanji KK, Alam MK, Gudipaneni RK, et al.: Do learning style preferences influence the cumulative gross point average and self-directed learning hours in dental students: a preliminary study. BMC Med. Educ. 2022; 22(1): 493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeitz G, Donker A, Parpala A: Studying in an innovative teaching-learning environment: design-based education at a university of applied sciences. Learn. Environ. Res. 2023; 1–19.\n\nGeorge T, Carey RA, Abraham OC, et al.: Trainee doctors in medicine prefer case-based learning compared to didactic teaching. J. Family Med. Prim. Care. 2020; 9(2): 580–584. PubMed Abstract \| Publisher Full Text\n\nvan der Graaf J , Lim L, Fan Y, et al.: The dynamics between self-regulated learning and learning outcomes: An exploratory approach and implications. Metacogn. Learn. 2022; 17(3): 745–771. Publisher Full Text\n\nHan F: Relations between Students’ Study Approaches, Perceptions of the Learning Environment, and Academic Achievement in Flipped Classroom Learning: Evidence from Self-Reported and Process Data. J. Educ. Comput. Res. 2023; 61(6): 1252–1274. Publisher Full Text\n\nHermida J: Facilitating deep learning: Pathways to success for university and college teachers. CRC Press; 2014.\n\nHongyu L, Hongxin Y: A review of research on children’s metacognitive development. Psychol. Behav. Res. 2004; 2(1): 383.\n\nHoule CO: The inquiring mind.1961.\n\nHsieh HM, Maritz A: Effects of Flipped Teaching on Entrepreneurship Professional Student Learning Motivation, Self-directed Learning, and Learning Outcome. Contemp. Educ. Technol. 2023; 15(4). Publisher Full Text\n\nHua M, Wang L: The impact of self-directed learning experience and course experience on learning satisfaction of university students in blended learning environments: the mediating role of deep and surface learning approach. Front. Psychol. 2024; 14: 1278827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen E, Nickelsen L: Deeper learning: 7 powerful strategies for in-depth and longer-lasting learning. Corwin Press; 2008.\n\nKenna T: Podcasting urban geographies: examining the utility of student-generated research podcasts for deep learning and education for sustainable development. J. Geogr. High. Educ. 2023; 47(4): 533–552. Publisher Full Text\n\nKnowles MS: Self-directed learning: A guide for learners and teachers. 1975.\n\nLahdenperä J, Rämö J, Postareff L: Contrasting undergraduate mathematics students’ approaches to learning and their interactions within two student-centered learning environments. Int. J. Math. Educ. Sci. Technol. 2023; 54(5): 687–705. Publisher Full Text\n\nLai C, Chen Q, Wang Y, et al.: Individual interest, self-regulation, and self-directed language learning with technology beyond the classroom. Br. J. Educ. Technol. 2024; 55(1): 379–397. Publisher Full Text\n\nLing H, Jiahou L: Promote students’ deep learning. Computer Teaching and Learning. Modern Teaching. 2005; 5: 29–30.\n\nLitzinger TA, Wise JC, Lee SH: Self-directed learning readiness among engineering undergraduate students. J. Eng. Educ. 2005; 94(2): 215–221. Publisher Full Text\n\nLong HB: Understanding self-direction in learning. Practice and theory in self-directed learning. 2000; pp. 11–24.\n\nLounsbury JW, Levy JJ, Park SH, et al.: An investigation of the construct validity of the personality trait of self-directed learning. Learn. Individ. Differ. 2009; 19(4): 411–418. Publisher Full Text\n\nMa X: Influence study of learners’ independent learning ability on learning performance in online learning. International Journal of Emerging Technologies in Learning (iJET). 2022; 17(9): 201–213. Publisher Full Text\n\nMarton F, Säljö R: On qualitative differences in learning: I—Outcome and process. Br. J. Educ. Psychol. 1976; 46(1): 4–11. Publisher Full Text\n\nMerriam SB, Baumgartner LM: Learning in adulthood: A comprehensive guide. John Wiley & Sons; 2020.\n\nMoonen-van Loon JM, Govaerts M, Donkers J, et al.: Toward automatic interpretation of narrative feedback in competency-based portfolios. IEEE Trans. Learn. Technol. 2022; 15(2): 179–189. Publisher Full Text\n\nNaidoo D: Deep learning opportunities in the geography classroom. S. Afr. J. Educ. 2021; 41(2): 1–10. Publisher Full Text\n\nNhat TNM, Van Le T: Effects of Reflective Thinking On Deep Learning in Theoretical Linguistics Classes. Int. J. Instr. 2023; 16(4): 101–120. Publisher Full Text\n\nOnah DF, Pang EL, Sinclair JE: Cognitive optimism of distinctive initiatives to foster self-directed and self-regulated learning skills: A comparative analysis of conventional and blended-learning in undergraduate studies. Educ. Inf. Technol. 2020; 25(5): 4365–4380. Publisher Full Text\n\nParpala A, Mattsson M, Herrmann KJ, et al.: Detecting the variability in student learning in different disciplines—a person-oriented approach. Scand. J. Educ. Res. 2022; 66(6): 1020–1037. Publisher Full Text\n\nPartanen LJ, Myyry L, Asikainen H: Physical chemistry students’ learning profiles and their relation to study-related burnout and perceptions of peer and self-assessment. Chem. Educ. Res. Pract. 2024; 25: 474–490. Publisher Full Text\n\nPereira AS, Wahi MM: Development and Testing of a Roleplaying Gamification Module to Enhance Deeper Learning of Case Studies in an Accelerated Online Management Theory Course. Online Learning. 2021; 25(3): 101–127. Publisher Full Text\n\nRui L: Prisma flowchart. [Data set]. Figshare. 2024a, June. Publisher Full Text\n\nRui L: The role of self-directed learning in promoting deep learning processes: a systematic literature review. [Data set]. Figshare. 2024b, June. Publisher Full Text\n\nRui L: Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. [Data set]. Figshare. 2024c, June. Publisher Full Text\n\nSalleh UKM, Zulnaidi H, Rahim SSA, et al.: Roles of self-directed learning and social networking sites in lifelong learning. Int. J. Instr. 2019; 12(4): 167–182. Publisher Full Text\n\nSaqr M, Matcha W, Jovanovic J, et al.: Transferring effective learning strategies across learning contexts matters A study in problem-based learning. Australas. J. Educ. Technol. 2023; 39(3): 35–57. Publisher Full Text\n\nSegú Odriozola MI: Autoethnography as a Tool for the Achievement of Deep Learning of University Students in Service-Learning Experiences. Soc. Sci. 2023; 12(7): 395. Publisher Full Text\n\nStephen JS, Rockinson-Szapkiw AJ: A high-impact practice for online students: the use of a first-semester seminar course to promote self-regulation, self-direction, online learning self-efficacy. Smart Learning Environments. 2021; 8(1): 6. Publisher Full Text\n\nSugden N, Brunton R, MacDonald J, et al.: Evaluating student engagement and deep learning in interactive online psychology learning activities. Australas. J. Educ. Technol. 2021; 37(2): 45–65. Publisher Full Text\n\nSun W, Hong JC, Dong Y, et al.: Self-directed learning predicts online learning engagement in higher education mediated by the perceived value of knowing learning goals. Asia Pac. Educ. Res. 2023; 32(3): 307–316. Publisher Full Text\n\nThompson AR, Lake LP: Relationship between learning approach, Bloom’s taxonomy, and student performance in an undergraduate Human Anatomy course. Adv. Health Sci. Educ. 2023; 28(4): 1115–1130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTough AM: The association was obtained by adult self-teachers. Adult Education (USA). 1966; 17: 33–37.\n\nTuononen T, Hyytinen H, Räisänen M, et al.: Metacognitive awareness about university students’ learning profiles. Metacogn. Learn. 2023; 18(1): 37–54. Publisher Full Text\n\nWeng C, Chen C, Ai X: A pedagogical study on promoting students’ deep learning through design-based learning. Int. J. Technol. Des. Educ. 2023; 33(4): 1653–1674. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiley K: Effects of a self-directed learning project and preference for structure on self-directed learning readiness. Nurs. Res. 1983; 32(3): 181–185. PubMed Abstract \| Publisher Full Text\n\nYağan SA, Parlar H: Investigating the Relationships Between University Students’ Epistemological Beliefs and Learning Approaches: A Path Analysis Study. SAGE Open. 2023; 13(2): 21582440231184855. Publisher Full Text\n\nYaguarema M, Zambrano RJ, Salavarría M: Analysis of the Deep Learning Strategies Questionnaire with Ecuadorian Students. Frontiers in Education. Frontiers Media SA; 2022, November; Vol. 7: 1004874. Publisher Full Text" }	[ { "id": "311630", "date": "21 Aug 2024", "name": "FX. Risang Baskara", "expertise": [ "Reviewer Expertise My research expertise spans several interconnected areas in educational technology and pedagogy", "with a focus on innovative approaches in higher education. I have extensive experience in blended learning and flipped classroom methodologies", "investigating their impact on student engagement and learning outcomes. My work in Computer Assisted Language Learning (CALL) explores the integration of technology in language acquisition. I also conduct research on the applications of Artificial Intelligence in education", "particularly in personalized learning and automated assessment. Additionally", "my background in Instructional Design and Technology informs my work on developing effective digital learning environments. These areas of research are underpinned by a strong foundation in Higher Education Pedagogy", "where I examine best practices for promoting deep learning and critical thinking skills in university settings." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examines the role of self-directed learning (SDL) in promoting deep learning processes among university students. The authors conducted a search of the Web of Science database for relevant literature published between January 2020 and March 2024. After screening, 39 papers were included in the final analysis. The review explores the interrelationship between SDL and deep learning, factors that facilitate deep learning in SDL environments, and how students experience and achieve deep learning through SDL. The authors conclude that SDL plays a crucial role in promoting deep learning by enhancing student engagement, motivation, and personalized learning. They identify several key conditions that facilitate deep learning in SDL contexts and describe strategies students use to achieve deep learning.\nMajor points that must be addressed:\nMethodology:\nProvide the full search strategy, including Boolean operators and any limits applied. Name and describe the specific quality assessment tool used to evaluate included studies. Provide more detail on the data extraction and synthesis process. Describe the thematic analysis approach in greater depth.\n\nResults presentation:\nInclude a clear PRISMA flow diagram showing the number of studies at each stage of screening. Provide a summary table of included studies with key characteristics (e.g. study design, sample size, key findings). Present results more systematically, clearly linking findings to specific included studies.\n\nCritical appraisal:\nInclude a more thorough critical appraisal of the included studies, discussing potential biases and limitations. Address heterogeneity among included studies in terms of designs, populations, and outcomes measured.\n\nConclusions:\nEnsure all conclusions are clearly supported by evidence presented in the results. Discuss limitations of the review itself and the overall quality of evidence. Provide more specific recommendations for future research to address gaps identified.\n\nAdditional suggestions for improvement:\nIntroduction:\nMore clearly define the specific gap in knowledge this review aims to address. Provide a stronger rationale for the chosen research questions.\n\nMethods:\nJustify the choice of date range for included studies (2020-2024). Explain why only one database (Web of Science) was searched.\n\nResults:\nProvide more quantitative summary of findings where possible (e.g. number of studies supporting key themes). Consider using more subheadings to improve organization and clarity.\n\nDiscussion:\nCompare findings more explicitly to previous reviews or theoretical frameworks in this area. Discuss potential implications for educational practice in more depth.\n\nOverall:\nImprove consistency in use of terminology (e.g. \"self-directed learning\" vs \"autonomous learning\"). Enhance readability by reducing length and eliminating redundancy in some sections.\n\nAddressing these points will significantly strengthen the scientific rigor and overall quality of this systematic review. The topic is important and timely, and with improvements to methodology and reporting, this review has the potential to make a valuable contribution to the literature on self-directed learning and deep learning in higher education.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] }, { "id": "337599", "date": "10 Dec 2024", "name": "Sukie Van Zyl", "expertise": [ "Reviewer Expertise Deeper self-directed learning", "Computer Science Education", "Cooperative learning" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the article seemed relevant, but the research questions do not provide a clear focus to the study. The concepts SDL and DL are used in the research questions, but unfortunately a sound understanding of these concepts are not indicated by the authors. It is assumed that SDL will lead to DL. Although SDL is a complex process, where several aspects have to be incorporated (Knowles, 1975) the research aimed to single out Individual factors or “SDL conditions” to lead to DL. A key research question, the interrelationship between SDL and DL is not clearly answered.\n\nThe concept deep learning (DL) is not sufficiently unpacked and argued. The term DL is often in literature used in the context of machine learning and the title could confuse the reader regarding the topic of discussion. The articles cited don’t specifically mention the concept deep learning, but rather refer to deep approaches to learning. Literature on deeper learning and deeper self-directed learning should also have been consulted to indicate a deep understanding of the literature. Although the methods section indicated that such research was consulted, a lack of evidence was found of such articles.\n\nSelf-directed learning is not adequately explained. Sweeping statements are made on Knowles’ stance on self-directed learning, adult learning and children. Self-directed learning is mentioned as a personality trait, but no sources are provided. Although Knowles' definition is mentioned, it is not used as a basis for SDL in the remainder of the chapter.\n\nThe theories (self-determination theory) and meta-cognitive theory are not applied as a basis for the data analysis and discussion. It is not clear which specific metacognitive theory the authors are referring to.\n\nDiscussion on SDL Inconsistent reference to SDL. Knowels’ definition was used as the basis for defining SDL as a process, but SDL is also referred to as a “learning approach' by the authors. Lack of critical engagement with literature and generalizations and conclusions are being made based on the findings of one or a few research studies. Example (Altinpulluk et al., 2023) specifically focused in the context of a MOOC, but the findings are generalized to other contexts. SDL should also be clearly distinguished from self-regulation. Page 4 Many concepts are mentioned, e.g. blended learning, learning styles, cognitive load. But only few literature are mentioned, and a one-sided perspective is presented without critically engaging in these concepts. Too many other fields are discussed and a lack of focus on the title and aim of the research. Not sufficiently indicated that SDL promotes deep learning and academic achievement. What is meant by academic achievement? Distinguish between deep learning and academic achievement .\nDiscussion on DL The term deep learning should be argued and clarified. I assume that you refer to 'deep approaches to learning', which is often mentioned in the articles that were cited, in stead of 'deep learning'. “7 Powerful strategies for deep learning” The title of the book is incorrect, it refers to “deeper learning” which holds fundamental implications for the arguments presented in this proposed article. Technology is viewed as the factor that promotes deep learning, not taking into account the role of the teaching and learning strategies in which technology is applied (ignoring the TPACK framework). Contradicting arguments are presented. For example it is mentioned that research is increasingly focusing on DL and autonomous learning, but then it is also argued that the integration of DL and SDL is relatively unexplored. The principles of deep learning have not clearly been explained. Should the process of SDL not rather be implemented in such a way to result in DL? Blended learning on its own should not be generalized as resulting in deep learning, because other 'factors' for developing deeper learning should be incorporated. Teacher-directed approaches can still be applied in class in the context of blended learning.\nResearch design Deep learning = 265,092 articles. What was done to exclude deep learning in the context of machine learning? Did these articles include “deeper learning” and “deep approaches to learning” that was also mentioned? Which process did the one researcher follow for examining all 265 092 articles? The number of articles indicated in figures 1 and 2 do not align with the numbers in the discussion of the literature screening process. It seems that the articles focused either on SDL (n=14) or on DL (n=25). How was it then possible to determine achievement of DL within SDL? What about articles that focused on both concepts, which indicated how DL can be developed by SDL or the role of SDL in fostering DL? It was stated that SDL leads to DL, but articles focused either on SDL or on DL. In my view the literature that were selected for this study was not sufficient for answering the research questions. It is stated that the literature was limited to January 1, 2020, and March 8, 2024, but several citations were out of the range. A more in-depth indication of the themes that emerged from the literature is needed. Technology is indicated as a key factor for DL, but the application of technology and the integration in sound pedagogical strategies are not mentioned. Aspects such as PBL, assessment, cognitive load are mentioned, without clearly linking it to the research problem. It is unclear why PBL and flipped classroom are focused on but ignoring other relevant strategies. Sweeping statements are often made, for example that social media will enhance critical thinking. Not clear why metacognitive skills and reflection and evaluation are separated under different headings (see p. 12).\nConclusion The statement made in the conclusion that students should be provided with an abundance of resources, is concerning, and contradicts the intention of SDL. The research questions were unfortunately not sufficiently answered in the conclusion section.\n\nGeneral\nSome long paragraphs are noticed and the argument line is difficult to follow. Lai et al. (2024). Shouldn’t this be a 2023 citation? Some resources are quoted out of context. They are for example mentioned with regards to DL, but then they don't refer to DL. The abbreviation SDL is not consistently used. Several tables that are mentioned are not 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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/13-761

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.